Ryegrass pollen allergen

ABSTRACT

The present invention provides a nucleic acid sequences coding for the ryegrass pollen allergens Lol pIa and Lol pIb, purified LoI pIa and Lol pIb protein and fragments thereof, methods of producing recombinant Lol pIa and Lol pIb or at least one fragment thereof or derivative or homologue thereof, and methods of using the nucleic acid sequences, proteins and peptides of the invention.

This application is a divisional of U.S. Ser. No. 07/746,703 filed onAug. 16, 1991, now abandoned, which Is a continuation-in-part of U.S.Ser. No. 07/585,086 filed Oct. 26, 1990, now abandoned, which claimspriority to Australian Patent Application No. PK 1823/90 filed Aug. 17,1990, all the disclosures of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the major allergenic protein Lol pIbfrom pollen of ryegrass, Lolium perenne L. and to derivatives andhomologues thereof and to allergenic proteins immunologically relatedthereto. The present invention is also directed to recombinant Lol pIaand Lol pIb and their derivatives and to expression vectors capable ofdirecting synthesis of same. Even more particularly, the presentinvention is directed to cDNA separately encoding Lol pIa and Lol pIband to expression vectors comprising same.

BACKGROUND OF THE INVENTION

Allergens constitute the most abundant proteins of grass pollen, whichis the major cause of allergic disease in temperate climates (Marsh(1975) Allergens and the genetics of allergy; in M. Sela (ed), TheAntigens, Vol. 3, pp 271-359, Academic Press Inc., London, New York).,Hill et al. (1979) Medical Journal of Australia 1, 426-429). The firstdescriptions of the allergenic proteins in ryegrass showed that they areimmunochemically distinct, and are known as groups I, II, III and IV(Johnson and March (1965) Nature, 206, 935- ; and Johnson and Marsh(1966) Immunochemistry 3, 91-100). Using the International Union ofImmunological Societies' (IUIS) nomenclature,,these allergens aredesignated Lol pI, Lol pIl, Lol pIII and Lol pIV.

These four proteins have been identified in pollen ryegrass, Loliumperenne L., which act as antigens in triggering immediate (Type 1)hypersensitivity in susceptible humans.

Lol pI is defined as an allergen because of its ability to bind tospecific IgE in sera of ryegrass-sensitive patients, to act as anantigen in IgG responses and to trigger T-cell responses. The allergenicproperties have been assessed by direct skin testing of grasspollen-sensitive patients. The results showed that 84% had a skinsensitivity to Lol pI (Freidhoff et al., (1986) J. Allergy Clin.Immunol. 78: 1190-1201) demonstrating the primary importance of thisprotein as the major allergen. Furthermore, 95% of patients demonstratedto be grass pollen-sensitive possessed specific IgE antibody that boundto Lol pI, as demonstrated by immunoblotting (Ford and Baldo (1986)International Archives of Allergy and Applied Immunology 81: 193-203).

Substantial allergenic cross-reactivity between grass pollens has beendemonstrated using an IgE-binding assay, the radioallergo-sorbent test(RAST), for example, as described by Marsh et al. (1970) J. Allergy, 46,107-121, and Lowenstein (1978) Prog. Allergy, 25, 1-62. (Karger, Basel).

The immunochemical relationship of Lol pI with other grass pollenantigens have been demonstrated using both polyclonal and monoclonalantibodies (e.g. Smart and Knox (1979) International Archives of Allergyand Applied Immunology 62: 173-187; Singh and Knox (1985) InternationalArchives of Allergy and Applied Immunology 78, 300-304). Antibodies havebeen prepared to both purified proteins and IgE-binding components.These data demonstrate that the major allergen present in pollen ofclosely related grasses is immunochemically similar to Lol pI (Singh andKnox, supra).

SUMMARY OF THE INVENTION

In accordance with the present invention, it has been discovered thatLol pI comprises two proteins, designated herein Lol pIa and Lol pIb.The genes encoding these proteins have now been cloned permitting thelarge scale production of the recombinant allergens. One aspect of thepresent invention thus provides nucleic acid sequences coding for LolpIa (SEQ ID NO: 3 and SEQ ID NO: 5) and Lol pIb (SEQ ID NO: 1).

Another aspect of the present invention relates to a recombinant vectorcomprising a DNA sequence encoding a protein displaying allergenicactivity from pollen of a grass species. More particularly, the grassspecies belongs to the family Poaceae (Gramineae), and even moreparticularly, to the genus Lolium. Still even more particularly, theallergenic protein in characterized as being immunologicallycross-reactive with antibody to Lol pIa or Lol pIb protein of Loliumperenne pollen, namely:

Pooid (festucoid) grasses. GROUP 1: Triticanea: Bromus inermis, smoothbrome; Agropyron revens, English couch; A.cristatum; Secale cereals ryeTriticum aestivum, wheat. GROUP 2: Poanae: Dactylis glomerata, orchardgrass of cocksfoot; Festuca elatior, meadow fescue; Lolium perenne,perennial ryegrass, L.multiflorum, Italian ryegrass; Poa pratensis,Kentucky bluegrass; P.compressa, flattened meadow grass; Avena sativa,oat; Holcus lanatus, velvet grass or Yorkshire fog; Anthoxanthumodoratum; sweet vernal grass; Arrhenatherum elatius, oat grass; Agrostisalba, red top; Phleum pratense, timothy; Phalaris arundinacea, reedcanary grass. Panicoid grass, Paspalum notatum, Bahia grass,Andropogonoid grasses: Sorghum halepensis, Johnson grass.

A further aspect of the present invention relates to a recombinantvector comprising a DNA sequence encoding the allergenic protein Lol pIaor Lol pIb of ryegrass, Lolium perenne , L. pollen, or derivatives orhomologues thereof. More particularly, the present invention relates toa recombinant DNA molecule comprising a eukaryotic or prokaryotic originof replication, a detectable marker, a DNA sequence encoding either LolpIa or Lol pIb allergenic protein or derivatives or homologues thereofor an allergenic protein cross-reactive with an antibody to said Lol pIaor Lol pIb protein or their derivatives or homologues and optionally apromoter sequence capable of directing transcription of said allergenicproteins.

Yet another aspect of the present invention contemplates a method forproducing recombinant Lol pIa or Lol pIb or derivatives or homologuesthereof or an allergenic protein immunologically reactive to antibodiesto Lol pIa or Lol pIb or a derivative or homologue thereof, comprisingculturing an organism containing a replicable recombinant DNA molecule,said molecule comprising a promoter capable of expression in saidorganism, the gene encoding Lol pIa or Lol pIb or their derivatives orhomologues or an immunologically related protein of Lol pIa or Lol pIblocated downstream of and transcribed from said promoter, a selectablemarker and a DNA vehicle containing a prokaryotic or eukaryotic originof replication, under conditions and for a time sufficient for saidrecombinant DNA molecule to be stably maintained and direct thesynthesis of Lol pIa or Lol pIb or their derivatives or homologues.

In yet another aspect of the present invention, there is providednon-native (i.e., recombinant or chemically synthesized) Lol pIa (SEQ IDNO: 5 and SEQ ID NO: 6) or Lol pIb (SEQ ID NO: 1 and SEQ ID NO: 2) ortheir derivatives or homologues or a non-native allergenic proteinimmunologically cross-reactive to antibodies to Lol pIa or Lol pIb ortheir derivatives or homologues.

The Lol pIa and Lol pIb proteins, and fragments or portions derivedtherefrom (peptides) can be used in methods of diagnosing, treating andpreventing allergic reactions to ryegrass pollen.

Still yet another aspect of the present invention relates to antibodiesto non-native Lol pIa or Lol pIb or a derivative of homologue thereof.

In still yet another aspect of the present invention, there is provideda method for detecting an antibody to an allergenic protein from pollenof the family Poaceae (Gramineae) in serum or other biological fluidcomprising contacting said serum or fluid with recombinant Lol pIa orLol pIb or their antigenic derivatives for a time and under conditionssufficient for an antibody-LolpIa or Lol-pIb domplex to form andsubjecting said complex to a detecting means.

Another aspect of the present invention relates to a recombinant DNAmolecule comprising a ryegrass pollen promoter sequence or homologue ordegenerate form thereof located on said molecule and further having oneor more restriction sites down stream of said promotor such that anucleotide sequence inserted into one or more of these sites istranscribable in the correct reading frame.

In one preferred embodiment, the recombinant DNA molecule comprises thepromoter directing synthesis of Lol pIa or Lol pIb from pollen ofryegrass, Lolium perenne L. and is thereby a developmentally regulated,pollen specific, expression vector.

A further aspect of the present invention contemplates a method forinducing nuclear male sterility in plants of the family Poaceaecomprising the steps of:

a) developing a plant carrying a recombinant DNA molecule comprising aryegrass pollen promoter sequence or homologue or degenerate formthereof located on said molecule and a nucleotide sequence encoding apolypeptide having a deleterious function in cells derived from thefamily Poaceae, said nucleotide sequence transcribable from saidpromoter, and said recombinant DNA molecule stably contained in pollenproducing cells, and,

b) growing said plants under conditions and for a time sufficient fortheir developmental stage to cause expression of said nucleotidesequence from said promoter thereby producing the polypeptide having adeleterious function on said pollen producing cells such that pollenformation is inhibited or said pollen is inactive.

Further features of the present invention will be better understood fromthe following detailed description of the preferred embodiments of theinvention in conjunction with the appended figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1a and 1 b shows isolation of cDNA clones specific for the PoaceaeGroup I allergens. FIG. 1a illustrates recognition of a positive clone(12R) by three different MAbs FMC-A1 (40.1), FMC-A7 (12.3), 3.2 (Kahn &Marsh (1986) Molec. Immunol. 23: 1281-1288; Singh & Knox (1985)International Archives of Allergy and Applied Immunology 78, 300-304;Smart et al. (1983) International Archives of Allergy and AppliedImmunology 72 243-248) and IgE from allergic patients' sera. C is thecontrol in which the primary MAb was omitted. FIG. 1b shows animmunoblot analysis of MAbs and IgE binding to group I antigens fromrye-grass pollen. Lane I shows total protein profile (Coomassie bluestaining); Lane 2: MAb 40.1; Lane 3: MAb 21.3; Lane 4: MAb 12.3; Lane 5:IgE antibodies.

FIG. 2 shows tissue-type and cell-type specific expression of group Iallergen transcripts. FIG. 2a shows RNA blot hybridization. Poly(A)+RNAs were isolated from different plant tissues: seed leaf, root andpollen. FIG. 2b shows immunoblot analysis of tissue-type and cell-typespecific distribution of group I antigens. The soluble proteins wereextracted from different plant tissues: flower, leaf root and pollen,and were immunoblotted using MAbs 40.1, 12.3 and IgE antibodies.

FIGS. 3a, 3 b and 3 c show the cDNA sequence (SEQ ID NO: 1), predictedamino acid sequence (SEQ ID NO: 1 and SEQ ID NO: 2) and hydrophilicityprofile of rye-grass pollen clone 12R. FIG. 3a shows a schematicrestriction map of lambda-12R cDNA. The hatched box represents thepredicted translation open reading frame. FIG. 3b shows the nucleotideand deduced amino acid sequence of the 1242 nucleotide EcoR1 cDNA insertlambda-12R (SEQ ID NO: 1). The deduced amino acid sequence representedby the single letter code is shown above the DNA sequence in FIG. 3b,and begins at the first potential in-frame initiation codon atnucleotide 40. One uninterrupted open reading frame continues for 308amino acids (numbered above the DNA sequence in FIG. 3b) and ends withthe TGA stop codon denoted by an asterisk. The putative signal peptideis indicated by negative numbers. The amino acid residues 1-9, 12-17,and 19 were identified by N-terminal sequencing. FIG. 3c shows thehydrophilicity profile of predicted amino acid sequence based on methodof Hopp and Woods (1981) Proc. Natl Acad. Sci. USA 78: 3824-3828, with awindow of seven amino acids.

FIG. 4 shows the delineation of IgE and MAb-reacting epitopes in Lol pIbclone 12R using immunoblotting: FIG. 4a: IgE antibodies; FIG. 4b, MAb40.1 and FIG. 4c, HAb 12.3. Controls for FIGS. 4a-c are provided bybacteria transformed with non-recombinant plasmids.

FIGS. 5a, 5 b and 5 c show detection of Lol pIa and Lol pIb in maturepollen of rye-grass using specific MAbs and immunogold probes. FIG. 5ashows whole pollen grains visualized by scanning electron microscopy,showing the single germinal pore. Scale bar, 30 um. FIG. 5b showsdetection of cellular sites of Lol pIa and Lol pIb by immuno-goldlocalization—double labelling. FIG. 5c shows the appearance of fresh,viable pollen after exposure to water for 30 s , dark fieldillumination.

FIG. 6 shows the nucleotide sequence and predicted amino acid sequenceof clone 13R which has a sequence coding for part of Lol pIa (SEQ ID NO:3). (The predicted amino acid sequence is also shown in SEQ ID NO: 4).

FIGS. 7a and 7 b show the nucleotide sequence of cDNA clone 26.j and itspredicted amino acid sequence (SEQ ID NO: 5). (The predicted amino acidsequence is also shown in SEQ ID NO: 6). Clone 26.j is a PCR-generated,full-length clone of Lol pIa.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided the genesencoding the ryegrass pollen allergens Lol pIa and Lol pIb, a method forexpressing same in a host cell, thereby providing a source ofrecombinant Lol pIa and Lol pIb and the promoter of the Lol pIa and LolpIb or any genetic sequence pIaced downstream thereof.

The data herein show that what was considered to be the major allergenof rye-grass pollen, Lol pI, actually comprises two different proteins:Lol pIa, a 35 kD protein, pI 5.5 and Lol pIb, a 31/33 kD protein, pI9.0. Complementary DNA clones encoding Lol pIa and Lol pIb have beenseparately isolated and characterized. Lol pIb has a different primarystructure and composition from Lol pIa, as deduced from cDNA cloning,NH₂-terminal amino acid sequence and the absence of allergeniccross-reactivity. Lol pIb is synthesized in pollen as a preallergen witha 25 amino acid signal peptide which targets the allergen to plastids.This is followed by cleavage of the peptide, and in mature pollen theallergen occurs predominantly in the starch grains.

The original source of the genetic material is fresh ryegrass pollenfrom Lolium perenne L., collected from field sources near Melbourne,Australia and bulk collected pollen from a supplier (Greer Laboratories,Lenoir, N.C.). These sources of pollen are not intended to limit thescope of the invention since they only represent one convenient supplyof the pollen. The present invention can be practiced using pollen fromany location.

“Gene”, is used, in respect of the present invention, in its broadestsense and refers to any contiguous sequence of nucleotides, thetranscription of which leads to an mRNA molecule, which mRNA molecule iscapable of being translated into a protein. The gene encoding Lol pIa orLol pIb means the nucleotide sequence encoding the proteins orderivatives or homologues of the proteins which may contain single ormultiple amino acid substitutions, deletions or additions includingderivatives containing the common antigenic epitope between Lol pIa andLol pIb. Similarly, in relation to the carbohydrate portion of Lol pIa,derivatives include single or multiple substitutions, deletions oradditions to said carbohydrate moiety. The Lol pIa and Lol pIb genesalso refer to cDNAs complementary to the uRNAs corresponding to the fullor partial length of the Lol pIa and Lol pIb proteins respectively.

It is expected that there are sequence polymorphisms in the nucleic acidsequence coding for Lol pIa and Lol pIb, and it will be appreciated byone skilled in the art that one or more nucleotides in the nucleic acid,sequences coding for Lol pIa and Lol pIb may vary among individual L.perenne plants due to natural allelic variation. Any and all suchnucleotide variations and resulting amino acid polymorphisms are withinthe scope of the invention. Polymorphisms of the gene coding for Lol pIadiscovered during sequencing of the gene are discussed in Example 9. Itmay also be appreciated by one skilled in the art that Lol p Ia and LolpIb may each be members of separate families of highly related geneswhose proteins are present in L. perenne pollen (e.g. Rafnar er al.(1991) J. Biol. Chem. 266: 1229-1236; Silvanovich et al. (1991) J. Biol.Chem. 266: 1204-1210). Nucleotide sequences and corresponding deducedamino acid sequences of any and all such related family members arewithin the scope of the present invention.

Accordingly, it is within the scope of the present invention toencompass Lol pIa or Lol pIb, at least one fragment (peptide) of Lol pIaor Lol pIb, and their amino acid and/or carbohydrate derivatives and toencompass nucleotide sequences, including DNA, cDNA and mRNA andhomologues or degenerate forms thereof, encoding Lol pIa or Lol pIb,said Lol pIa or Lol pIb fragments, or said derivatives thereof. It isfurther in accordance with the present invention to include moleculessuch as polypeptides fused to Lol pIa or Lol pIb, or at least one LolpIa or Lol pIb fragment, or their derivatives or to nucleotide sequencescontiguous to Lol pIa or Lol pIb, Lol pIa or Lol pIb fragment, and/orderivative-encoding nucleotide sequences. For example, for some aspectsof the present invention, it is desirable to produce a fusion proteincomprising Lol pIa, or Lol pIb or at least one fragment of Lol pIa orLol pIb, or their derivatives and an amino acid sequence from anotherpeptide or protein, examples of the latter being enzymes such asbeta-galactosidase, phosphatase, urease and the like. Most fusionproteins are formed by the expression of a recombinant gene in which twocoding sequences have been joined together such that their readingframes are in phase. Alternatively, proteins or peptides can be linkedin vitro by chemical means. All such fusion protein or hybrid geneticderivatives of Lol pIa or Lol pIb or their encoding nucleotide sequencesare encompassed by the present invention. Furthermore, by homologues andderivatives of Lol pIa or Lol pIb are meant to include syntheticderivatives thereof. The nucleotide sequences as elucidated herein, canbe used to chemically synthesize the entire proteins or generate anynumber of fragments (peptides) by chemical synthesis by well knownmethods (eg solid phase synthesis). All such chemically synthesizedpeptides are encompassed by the present invention. Accordingly, thepresent invention extends to isolated Lol pIa and Lol pIb, fragmentsthereof and their derivatives, homologues and immunological relativesmade by recombinant means or by chemical synthesis and may includederivatives containing the common antigenic epitope between Lol pIa andLol pIb. The terms isolated and purified are used interchangeably hereinand refer to peptides, protein, protein fragments, and nucleic acidsequences substantially free of cellular material or culture medium whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when synthesized chemically. Furthermore, the presentinvention extends to proteins or fragments (peptides) corresponding inwhole or part to the nucleotide coding sequence given in FIGS. 3b (SEQID NO: 1), 6 (SEQ ID NO: 3) and 7 (SEQ ID NO: 5), or to degenerate orhomologue forms thereof.

Fragments of nucleic acid within the scope of the invention includethose coding for parts of Lol pIa or Lol pIb that elicit an antigenicresponse in mammals, preferably humans, such as the stimulation ofminimal amounts of IgE;,the eliciting of IgG and IgM antibodies; or theeliciting of a T cell response such as proliferation and/or lymphokinesecretion and/or the induction of T cell anergy. The foregoing fragmentsof Lol pIa or Lol pIb are referred to herein as antigenic, fragments.Fragments within the scope of the invention also include those capableof hybridizing with nucleic acid from other plant species for use inscreening protocols to detect allergens that are cross-reactive with LolpIa or Lol pIb. As used herein, a fragment of the nucleic acid sequencecoding for Lol pIa or Lol pIb refers to a nucleotide sequence havingfewer bases than the nucleotide sequence coding for the entire aminoacid sequence of Lol pIa or Lol pIb and/or mature Lol pIa or Lol pIb.Generally, the nucleic acid sequence coding for the fragment orfragments of Lol pIa or Lol pIb will be selected from the bases codingfor the mature protein, however, in some instances it may be desirableto select all or a part of a fragment or A fragments from the leadersequence portion of the nucleic acid sequence of the invention. Thenucleic acid sequence of the invention may also contain linkersequences, restriction endonuclease sites and other sequences useful forcloning, expression or purification of Lol pIa or Lol pIb or fragmentsthereof.

Fragments of an allergen from ryegrass pollen, preferably Lol pIa or LolpIb, eliciting a desired antigenic response (referred to herein asantigenic fragments) may be obtained, for example, by screening peptidesproduced by recombinant methods from the corresponding fragment of thenucleic acid sequence of the invention coding for such peptides orsynthesized chemically using techniques known in the art. The peptidefragments of the allergen may be obtained by any method known in the artsuch as chemical cleavage of the allergen, arbitrary division of theallergen into fragments of a desired length with no overlap of thepeptides, or preferably division of the allergen into overlappingfragments of a desired length. The fragments are tested to determinetheir antigenicity and allergenicity. Fragments of Lol pIa or Lol pIbwhich are capable of eliciting a T cell response such as stimulation(i.e., proliferation or lymphokine secretion) and/or are capable ofinducing T cell anergy are particularly desirable. Fragments of Lol pIaor Lol pIb which do not bind immunoglobulin E (IgE) and/or which haveminimal IgE stimulating activity are also desirable. If the fragment orfragments of Lol pIa or Lol pIb bind IgE, it is preferable that suchbinding does not lead to histamine release, e.g., such binding does notcause cross-linking of IgE on mast cells. Minimal IgE stimulatingactivity refers to IgE stimulating activity that is less than the amountof IgE production stimulated by the whole Lol pIa or Lol pIb protein.Preferred fragments also include antigenic fragments which, whenadministered to a ryegrass pollen-sensitive individual, are capable ofmodifying the allergic response to ryegrass pollen of the individual,and antigenic fragments which, when administered to a ryegrasspollen-sensitive individual, are capable of modifying B-cell response,T-cell response or both B-cell and T-cell response of the individual toa ryegrass pollen antigen.

Screening for IgE binding to the protein or fragments thereof may beperformed by scratch tests or intradermal skin tests on laboratoryanimals or human volunteers, or in in vitro systems such as RAST(radioallergosorbent test), RAST inhibition, ELISA assay orradioimmunoassay (RIA).

The present invention provides expression vectors and host cellstransformed to express the nucleic acid sequences of the invention.Nucleic acid coding for Lol pIa or Lol pIb, or at least one fragmentthereof may be expressed in bacterial cells such as E. coli, insectcells, yeast, or mammalian cells such as Chinese hamster ovary cells(CHO). Suitable expression vectors, promoters, enhancers, and otherexpression control elements may be found in Sambrook et al. MolecularCloning: A Laboratory Manual, second edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. Expression in yeast,insect or mammalian cells would lead to partial or completeglycosylation of the recombinant material and formation of any inter- orintra-chain disulfide bonds, if such exist. Suitable vectors forexpression in yeast include YepSecl (Baldari et al. (1987) Embo J. 6:229-234); pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943); andJRY88 (Schultz et al. (1987) Gene 54: 113-123).

For expression in E. coli, suitable expression vectors include pTRC(Amann et al. (1988) Gene 69: 301-315); pGEX (Amrad Corp., Melbourne,Australia); pMAL (N.E. Biolabs, Beverly, Mass.); pRIT5 (Pharmacia,Piscataway, N.J.); and pSEM (Knapp et al. (1990) BioTechniques 8:280-281). he use of pTRC and PGEX will lead to the expression of unfusedprotein. The use of pMAL, pRIT5 and pSEM will lead to the expression ofallergen fused to maltose E binding protein (pMAL), protein A (pRIT5),or truncated β-galactosidase (PSEM). When Lol pIa or Lol pIb, fragment,or fragments thereof is expressed as a fusion protein, it isparticularly advantageous to introduce an enzymatic cleavage site at thefusion junction between the carrier protein and Lol pIa or Lol pIb orfragment thereof. Lol pIa or Lol pIb or fragment thereof may then berecovered from the fusion protein through enzymatic cleavage at theenzymatic site and biochemical purification using conventionaltechniques for purification of proteins and peptides. Suitable enzymaticcleavage sites include those for blood clotting Factor X or thrombin forwhich the appropriate enzymes and protocols for cleavage arecommercially available from for example Sigma Chemical Company, St.Louis, Mo. and N.E. Biolabs, Beverly, Mass.

Host cells can be transformed to express the nucleic acid sequences ofthe invention using conventional techniques such as calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,or electroporation. Suitable methods for transforming the host cells maybe found in Sambrook et al. supra, and other laboratory textbooks. Thenucleic acid sequences of the invention may also be synthesized usingstandard techniques.

Using the structural information now available, it is possible to designLol pIa or Lol pIb peptides which, when administered to a ryegrasspollen sensitive individual in sufficient quantities, will modify theindividual's allergic response to ryegrass pollen. This can be done, forexample, by examining the structure of Lol pIa (SEQ ID NO: 4 or SEQ IDNO: 6) or Lol pIb (SEQ ID NO: 2), producing peptides (via an expressionsystem, synthetically or otherwise) to be examined for their ability toinfluence B-cell and/or T-cell responses in ryegrass pollen sensitiveindividuals and selecting appropriate epitopes recognized by the cells.In referring to an epitope, the epitope will be the basic element orsmallest unit of recognition by a receptor, particularlyimmunoglobulins, histocompatibility antigens and T cell receptors wherethe amino acids essential to the receptor recognition may be contiguousand/or non-contiguous in the amino acid sequence. Amino acid sequenceswhich mimic those of the epitopes and which are capable of downregulating allergic response to Lol pIa or Lol pIb can also be used.

It is now also possible to design an agent or a drug capable of blockingor inhibiting the ability of ryegrass pollen allergen to induce anallergic reaction in ryegrass pollen sensitive individuals. Such agentscould be designed, for example, in such a manner that they would bind torelevant anti-Lol pIa or Lol pIb-IgE's, thus preventing IgE-allergenbinding and subsequent mast cell degranulation. Alternatively, suchagents could bind to cellular components of the immune system, resultingin suppression or desensitization of the allergic response to L. perennepollen allergens. A non-restrictive example of this is the use ofappropriate B- and T-cell epitope peptides, or modifications thereof,based on the cDNA/protein structures of the present invention tosuppress the allergic response to ryegrass pollen. This can be carriedout by defining the structures of B- and T-cell epitope peptides whichaffect B- and T-cell function in in vitro studies with blood componentsfrom ryegrass pollen sensitive individuals.

Protein, peptides or antibodies of the present invention can also beused for detecting and diagnosing ryegrass pollinosis. For example, thiscould be done by combining blood or blood products obtained from anindividual to be assessed for sensitivity to ryegrass pollen with anisolated antigenic peptide or peptides of Lol pIa or Lol pIb, orisolated Lol pIa or Lol pIb protein, under conditions appropriate forbinding of components (e.g., antibodies, T-cells, B-cells) in the bloodwith the peptide(s) or protein and determining the extent to which suchbinding occurs.

Additionally, sensitivity of a mammal to ryegrass pollen may bedetermined by administering to a mammal a sufficient quantity of theryegrass pollen allergen Lol pIa or Lol pIb, or at least one antigenicfragment thereof, produced in a host cell transformed with the nucleicacid sequence of Lol pIa or Lol pIb or fragment thereof or chemicallysynthesized, to provoke an allergic response in the mammal anddetermining the occurrence of an allergic response in the mammal to theryegrass pollen allergen.

The DNA used in any embodiment of this invention can be cDNA obtained asdescribed herein, or alternatively, can be any oligodeoxynucleotidesequence having all or a portion of a sequence represented herein, ortheir functional equivalents. Such oligodeoxynucleotide sequences can beproduced chemically or mechanically, using known techniques. Afunctional equivalent of an oligonucleotide sequence is one which is 1)a sequence capable of hybridizing to a complementary oligonucleotide towhich the sequence (or corresponding sequence portions) of SEQ ID NO:1), the sequence of SEQ ID NO: 3 or the sequence of SEQ ID NO: 5 orfragments thereof hybridizes, or 2) the sequence (or correspondingsequence portion) complementary to the sequence SEQ ID NO: 1, thesequence of SEQ ID NO: 3, or the sequence of SEQ ID NO: 5 and/or 3) asequence which encodes a product (e.g., a polypeptide or peptide) havingthe same functional characteristics of the product encoded by thesequence (or corresponding sequence portion) of SEQ ID NO: 1, thesequence of SEQ ID NO: 3 or the sequence of SEQ ID NO: 5. Whether afunctional equivalent must meet one or both criteria will depend on itsuse (e.g., if it is to be used only as an oligoprobe, it need meet onlythe first or second criteria and if it is to be used to produce a 1,1pIa or Lol pIb protein, it need only meet the third criterion).

It is also within the scope of the present invention to includeallergenic proteins immunologically cross-reactive with antibodies toLol pIa or Lol pIb or fragments thereof or their derivatives orhomologues and fragments of these allergenic proteins. “Immunologicallycross-reactive” is used in its broadest sense and refers generally to aprotein capable of detectable binding to an antibody, the latter beingspecific to Lol pIa or Lol pIb, or to fragments thereof or toderivatives or homologues of Lol pIa or Lol pIb or fragments thereof.Such an immunologically related protein is referred to herein as aimmunological relative to Lol pIa or Lol pIb.

Work by others has shown that high doses of allergens generally producethe best results (i.e., best symptom relief). However, many people areunable to tolerate large doses of allergens because of allergicreactions to the allergens. Modification of naturally-occurringallergens can be designed in such a manner that modified peptides ormodified allergens which have the same or enhanced therapeuticproperties as the corresponding naturally-occurring allergen but havereduced side effects (especially anaphylactic reactions) can beproduced. These can be, for example, a protein or peptide of the presentinvention (e.g., one having all or a portion of the amino acid sequenceof Lol pIa or Lol pIb), or a modified protein or peptide, or protein orpeptide analogue (e.g., a protein or peptide in which the amino acidsequence has been altered, such as by amino acid substitution, deletion,or addition, to modify immunogenicity and/or reduce allergenicity or towhich a component has been added for the same purpose). For example, LolpIa or Lol pIb protein or peptides can be modified using thepolyethylene glycol method of A. Sehon and co-workers. Wie et al. (1981)Int. Arch. Allergy Appl. Immunology. 64: 84-99.

Modification of Lol pIa or Lol pIb protein or peptides can also includereduction/alkylation (Tarr [1986) in: Methods of ProteinMicrocharacterization, J. E. Silver, ed. Humana Press, Clifton, N.J., pp155-194); acylation (Tarr, supra); esterification (Tarr, supra);chemical coupling to an appropriate carrier (Nishell and Shiigi, eds,(1980] Selected Methods in Cellular Immunology, WH Freeman, SanFrancisco, Calif.; U. S. Pat. No. 4,939,239); or mild formalin treatment(Marsh [1971] Int. Arch. Allergy Appl. Immunol. 41: 199-215).

The cloning of the cDNAs encoding Lol pIa and Lol pIb was based on therecognition of the protein expressed by Escherichia coli transformedwith lambda-gt 11 phage, using both specific monoclonal antibodies andspecific serum IgE from grass pollen-sensitive patients. Two such clonesare designated 12R and 13R. Also, monoclonal antibodies used were MAbs3.2, FMC A7 (12.3), 21.3 and FMC A1 (40.1) (Kahn & Marsh (1986) Molec.Immunol. 23: 1281-1288; Singh & Knox (1985) International Archives ofAllergy and Applied Immunology 78, 300-304; Smart et al. (1983)International Archives of Allergy and Applied Immunology 72 243-248).

Details of the cloning of Lol pIa and Lol pIb are given in the Examples.

The allergenic nature of the subject proteins are characterized in part,by their binding of the reaginic IgE antibodies which are present athigh levels in sera of allergic patients. The IgE binding to theepitopes on allergic proteins can be tested in the chromogenic assay inwhich allergens immobilized on a solid support can be visualized bysequential incubation in (1) allergic patients serum; (2)enzyme-labelled anti-IgE antibodies.

A variety of expression vectors can be constructed for the production ofLol pIa or Lol pIb or their derivatives. Accordingly, another aspect ofthe present invention contemplates a method of producing recombinant LolpIa or Lol pIb, or at least one fragment of Lol pIa or Lol pIb, or theirderivatives or homologues or their immunological relatives (ashereinbefore defined) comprising culturing an organism containing areplicable recombinant DNA molecule, said molecule comprising a promotercapable of expression in said organism, the Lol pIa or Lol pIb gene, atleast one fragment of Lol pIa or Lol pIb, or genes encoding theirderivatives, homologues or immunological relatives thereof, locateddownstream of and transcribed from said promoter, a selectable markerand a DNA vehicle containing a prokaryotic or eukaryotic origin ofreplication, under conditions and for a time sufficient and direct thesynthesis of Lol pIa or Lol pIb, at least one fragment of Lol pIa or LolpIb, or their derivatives, homologues or immunological relatives andthen isolating same.

The present invention also extends to the promoter of ryegrass pollenproteins, and particularly, to the promoter of the Lol pIa or Lol pIbgene. This promoter developmentally regulates Lol pIa or pIb geneexpression and is organ, i.e., pollen specific. Developmental regulationas used herein refers to the expression of a particular trait, in thiscase allergenic proteins in pollen, during a certain stage in a plantslife cycle and non-expression during another stage. Hence, the Lol pIaor Lol pIb promoter is particularly useful in allowing expression of LolpIa or Lol pIb, or any other gene or nucleotide sequence relativethereto, only during the development of pollen. The skilled artisan willimmediately recognize the importance of such promoters in selectivelyexpressing a particular trait during pollen formation.

Accordingly, the present invention contemplates a method of inhibitingpollen development or function and thereby inducing nuclear malesterility in plants of the family Poaceae, and in particular Loliumperenne L., comprising the steps of:

a) developing a plant carrying a recombinant DNA molecule comprising theryegrass pollen promoter sequence or homologue or degenerate formthereof located on said molecule and a nucleotide sequence encoding apolypeptide having a deleterious function in cells derived from thefamily Poaceae, said nucleotide sequence transcribable from saidpromoter, and said recombinant DNA molecule stably contained in pollenproducing cells, and,

b) growing said plants under conditions and for a time sufficient fortheir development stage to cause expression of said nucleotide sequencefrom said promoter thereby producing the polypeptide having adeleterious function on said pollen producing cells such that pollenformation is inhibited or said pollen is inactive.

Well established methods exist for introducing recombinant DNA moleculesinto plant cells such as use of Agrobacterium plasmids andelectroporation amongst others. By “deleterious function” in respect ofa polypeptide refers to a feature of said polypeptide that will inhibitcell growth, cause lysis of a cell, or inhibit various functions in acell and thereby prevent the normal functioning of the cell. In thiscase, lethal gene constructs having a deleterious function arecontemplated which inhibit or prevent pollen formation and therebyresult in a male sterile plant. Such “lethal genes” may encode enzymes,enzyme inhibitors, and/or toxic polypeptides, amongst other molecules.Alternatively, the lethal gene may encode an antisense RNA capable ofinhibiting translation of a particular species of mRNA, the translatedproduct thereof, being vital for pollen development.

Male sterile plants are particularly useful in developing hybrid cropvarieties.

The Lol pIa or Lol pIb promoter is isolatable from ryegrass genomic DNAby any number of procedures including use of promoter probes vectors,“chromosome walking” and S1 nuclease mapping and sequencing as DNAupstream of the transcription initiation site.

Accordingly, the present invention contemplates a recombinant DNAmolecule comprising a ryegrass pollen promoter sequence, and inparticular the promoter for the Lol pIa or Lol pIb gene, or homologuesor degenerate forms thereof located on said molecule and further havingone or more restriction endonuclease sites downstream of said promotersuch that nucleotide sequence inserted into one or more of these sitesis transcribable in the correct reading frame. As used herein, the“correct reading frame” has the same meaning as “in phase”. Theaforementioned DNA molecule will preferably also have a selectablemarker thereon, such as an antibiotic or other drug resistance gene,such as for example gene encoding resistance to ampicillin,carbenicillin, tetracycline, streptomycin and the like. The recombinantmolecule will further comprise a means for stable inheritance in aprokaryotic and/or eukaryotic cell. This can be accomplished by saidrecombinant molecule carrying a eukaryotic and/or a prokaryotic originof replication as hereinbefore described in relation to expressionvectors.

Alternatively, the recombinant molecule will carry a means forintegration into a host cell genome thereby permitting replication ofsaid recombinant molecule in synchrony with the replication of said hostcell genome. Examples of preferred prokaryotic hosts include cells E.coli, Bacillus and Pseudomonas amongst others. Preferred eukaryotichosts include cells from yeast and fungi, insects, mammals and plants.Even more preferred host cells are plants of the family Poaceae, and inparticular of the genus Lolium, such as Lolium perenne. Accordingly in apreferred embodiment, the Lol pIa or Lol pIb gene promoter with a geneencoding a deleterious function positioned relative thereto will becarried by a recombinant DNA molecule capable of integration into thegenome of cells of plants from the family Poaceae, or perenne. Such arecombinant DNA molecule is transferred to the aforementioned cells by,for example, electroporation. Ideally, said cells are callus-derivedcells. Said callus-derived cells transformed with said recombinant DNAmolecule are then permitted to regenerate into whole plants. Wholeplants entering the pollen of the Lol pIa or Lol pIb gene promoter and,hence, expression of the gene encoding a deleterious function.Consequently, pollen development is inhibited or prevented and a nuclearmale sterile plant results therefrom.

Alternatively, the Lol pIa or Lol pIb promoter will direct expression ofa gene having advantageous functions, such as a cytokinin. All suchrecombinant DNA molecules are encompassed by the present invention.

The present invention extends to monoclonal and polyclonal antibodies torecombinant or chemically synthesized Lol pIa or Lol pIb, or at leastone fragment of Lol pIa or Lol pIb, produced according to the methodsdescribed in International Patent Application No. PCT/AU89/00123 and totheir use in immunoassays and test kits as described therein.

The monoclonal antibodies used in the present work to screen the cDNAlibrary for Lol pIa clones showed cross-reactivity with allergenicproteins from pollen of various related grass species. This shows thereis a homology between allergenic proteins produced by these pollens withLol pI allergen supporting the applicability of the present invention toall related grasses. The present invention also relates to antibodies torecombinant Lol pIa or Lol pIb, their derivatives, homologues andimmunological relatives including their chemical synthetic derivatives.In the following discussion, reference to Lol pIa or Lol pIb includestheir derivatives, homologues and immunological relatives and chemicalsynthetic derivatives thereof. Such antibodies are contemplated to beuseful in developing detection assays (immunoassays) for said Lol pIa orLol pIb especially during the monitoring of a therapeutic or diagnosticregimen and in the purification of Lol pIa or Lol pIb. The antibodiesmay be monoclonal or polyclonal. Additionally, it is within the scope ofthis invention to include any second antibodies (monoclonal orpolyclonal) directed to the first antibodies discussed above. Thepresent invention further contemplates use of these first or secondantibodies in detection assays and, for example, in monitoring theeffect of a diagnostic or an administered pharmaceutical preparation.Furthermore, it is within the scope of the present invention to includeantibodies to the glycosylated regions of Lol pIa (where present), andto any molecules complexed with said Lol pIa. Accordingly, an antibodyto Lol pIa or Lol pIb encompasses antibodies to Lol pIa or Lol pIb, orantigenic parts thereof, and to any associated molecules (e.g.,glycosylated regions, lipid regions, carrier molecules, fused proteins,and the like).

The Lol pIa or Lol pIb, or parts thereof, considered herein are purifiedthen utilized in antibody production. Both polyclonal and monoclonalantibodies are obtainable by immunization with Lol pIa or Lol pIb, andeither type is utilizable for immunoassays. The methods of obtainingboth types of sera are well known in the art. Polyclonal sera are lesspreferred but are relatively easily prepared by injection of a suitablelaboratory animal with an effective amount of the purified Lol pIa orLol pIb, or antigenic parts thereof, collecting serum from the animal,and isolating specific sera by any of the known immunoadsorbenttechniques. Although antibodies produced by this method are utilizablein virtually any type of immunoassay, they are generally less favoredbecause of the potential heterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is particularlypreferred because of the ability to produce them in large quantities andthe homogeneity of the product. The preparation of hybridoma cell linesfor monoclonal antibody production derived by fusing an immortal cellline and lymphocytes sensitized against the immunogenic preparation canbe done by techniques which are well known to those who are skilled inthe art. (See, for example, Kohler and Milstein (1975) Nature256:495-497 and Kohler and Milstein (1986) Eur J. Immunol. 6:511-119).

Unlike preparation of polyclonal sera, the choice of animal is dependenton the availability of appropriate immortal lines capable of fusing withlymphocytes. Mouse and rat have been the animals of choice in hybridomatechnology and are preferably used. Humans can also be utilized assources for sensitized lymphocytes if appropriate immortalized human (ornonhuman) cell lines are available. For the purpose of the presentinvention, the animal of choice may be injected with from about 0.1 mgto about 20 mg of the purified Lol pIa or Lol pIb, or parts thereof.Usually the injecting material is emulsified in Freund's completeadjuvant. Boosting injections may also be required. The detection ofantibody production can be carried out by testing the antisera withappropriately labelled antigen. Lymphocytes can be obtained by removingthe spleen or lymph nodes of sensitized animals in a sterile fashion andcarrying out fusion. Alternatively, lymphocytes can be stimulated orimmunized in vitro, as described, for example, in Reading (1982) J.Immunol. Methods 53:261-291).

A number of cell lines suitable for fusion have been developed, and thechoice of any particular line for hybridization protocols is directed byany one of a number of criteria such as speed, uniformity of growthcharacteristics, deficiency of its metabolism for a component of thegrowth medium, and potential for good fusion frequency.

Intraspecies hybrids, particularly between like strains, work betterthan interspecies fusions. Several cell lines are available, includingmutants selected for the loss of ability to secrete myelomaimmunoglobin.

Cell fusion can be induced either by virus, such as Epstein-Barr orSendai virus, or polyethylene glycol. Polyethylene glycol (PEG) is themost efficacious agent for the fusion of mammalian somatic cells. PEGitself may be toxic for cells, and various concentrations should betested for effects on viability before attempting fusion. The molecularweight range of PEG may be varied from 1000 to 6000. It gives bestresults when diluted to from about 20% to about 70% (w/w) in saline orserum-free medium. Exposure to PEG at 37° C. for about 30 seconds ispreferred in the present case, utilizing murine cells. Extremes oftemperature (i.e., about 45° C.) are avoided, and preincubation of eachcomponent of the fusion system at 37° C. prior to fusion can be useful.The ratio between lymphocytes and malignant cells is optimized to avoidcell fusion among spleen cells and a range of from about 1:1 to about1:10 is commonly used.

The successfully fused cells can be separated from the myeloma line byany technique known by the art. The most common and preferred method isto chose a malignant line which is hypoxanthine Guanine PhosphoribosylTransferae (HGPRT) deficient, which will not grow in anaminopterin-containing medium used to allow only growth of hybrids, andaminopterin-containing medium used to allow only growth of hybrids andwhich is generally composed of hypoxanthine 1.10⁻⁴M, aminopterin1×10⁻⁵M, and thymidine 3×10⁻⁵, commonly known as the HAT medium. Thefusion mixture can be grown in the HAT-containing culture mediumimmediately after the fusion or 24 hours later. The feeding schedulesusually entail maintenance in HAT medium for two weeks and then feedingwith either regular culture medium or hypoxanthine, thymidine-containingmedium.

The growing colonies are then tested for the presence of antibodies thatrecognize the antigenic preparation. Detection of hybridoma antibodiescan be performed using an assay where the antigen is bound to a solidsupport and allowed to react to hybridoma supernatants containingputative antibodies. The presence of antibodies may be detected by“sandwich” techniques using a variety of indicators. Most of the commonmethods are sufficiently sensitive for use in the range of antibodyconcentrations secreted during hybrid growth.

Cloning of hybrids can be carried out after 21-23 days of cell growth inselected medium. Cloning can be preformed by cell limiting dilution influid phase or by directly selecting single cells growing in semi-solidagarose. For limiting dilution, cell suspensions are diluted serially toyield a statistical probability of having only one cell per well. Forthe agarose technique, hybrids are seeded in a semisolid upper layer,over a lower layer containing feeder cells. The colonies from the upperlayer may be picked up and eventually transferred to wells.

Antibody-secreting hybrids can be grown in various tissue cultureflasks, yielding supernatants with variable concentrations ofantibodies. In order to obtain higher concentrations, hybrids may betransferred into animals to obtain inflammatory ascites.Antibody-containing ascites can be harvested 8-12 days afterintraperitoneal injection. The ascites contain a higher concentration ofantibodies but include both monoclonals and immunglobulins from theinflammatory ascites. Antibody purification may then be achieved by, forexample, affinity chromatography.

The presence of Lol pIa or Lol pIb contemplated herein, or antibodiesspecific for same, in a patient's serum, plant or mammalian tissue ortissue extract, can be detected utilizing antibodies prepared as above,either monoclonal or polyclonal, in virtually any type of immunoassay. Awide range of immunoassay techniques are available as can be seen byreference to U.S. Pat. No. 4,015,043, 4,424,279 and 4,018,653. This, ofcourse, includes both single-site and two-site, or “sandwich”, assays ofthe non-competitive types, as well as in the traditional competitivebinding assays. Sandwich assays are among the most useful and commonlyused assays and are favored for use in the present invention. A numberof variations of the sandwich assay technique exist, and all areintended to be encompassed by the present invention. Briefly, in atypical forward assay, an unlabelled antibody is immobilized in a solidsubstrate and the sample to be tested brought into contact with thebound molecule. After a suitable period of incubation, for a period oftime sufficient to allow formation of an antibody-antigen secondarycomplex, a second antibody, labelled with a reporter molecule capable ofproducing a detectable signal is then added and incubated, allowing timesufficient for the formation of a tertiary complex ofantibody-antigen-labelled antibody (e.g., antibody-Lol pIb-antibody orantibody-Lol pIb-antibody). Any unreacted material is washed away, andthe presence of the antigen is determined by observation of a signalproduced by the reporter molecule. The results may either bequalitative, by simple observation of the visible signal, or may bequantitated by comparing with a control sample containing known amountsof hapten. Variations on the forward assay include a simultaneous assay,in which both sample and labelled antibody are added simultaneously tothe bound antibody, or a reverse assay in which the labelled antibodyand sample to be tested are first combined, incubated and then addedsimultaneously to the bound antibody. These techniques are well known tothose skilled in the art, including any minor variations as will bereadily apparent.

Although the following discussion is concerned with detecting Lol pIa orLol pIb, it is equally applicable to detecting antibodies to Lol pIa orLol pIb and it is intended to be sufficient description thereof. In thetypical forward sandwich assay, a first antibody having specificity forLol pIa or Lol pIb, or antigenic parts thereof, contemplated in thisinvention, is either covalently or passively bound to a solid surface.The solid surface is typically glass or a polymer, the most commonlyused polymers being cellulose, polyacrylamide, nylon, polystyrene,polyvinyl chloride or polypropylene. The solid supports may be in theform of tubes, beads, discs of microplates, or any other surfacesuitable for conducting an immunoassay. The binding processes arewell-known in the art and generally consist of cross-linking covalentlybinding or physically adsorbing, the polymer-antibody complex is washedin preparation for the test sample. An aliquot of the sample to betested is then added to the solid phase complex and incubated at 25° C.for a period of time sufficient to allow binding of any subunit presentin the antibody. The incubation period will vary but will generally bein the range of about 2-40 minutes. Following the incubation period, theantibody subunit solid phase is washed and dried and incubated with asecond antibody specific for a portion of the hapten. The secondantibody is linked to a reporter molecule which is used to indicate thebinding of the second antibody to the hapten.

By “reporter molecule,” as used in the present specification, is meant amolecule which, by its chemical nature, provides an analyticallyidentifiable signal which allows the detection of antigen-boundantibody. Detection may be either qualitative or quantitative. The mostcommonly used reporter molecules in this type of assay are eitherenzymes, fluorophores or radionuclide containing molecules (i.e.,radioisotopes). In the case of an enzyme immunoassay, an enzyme isconjugated to the second antibody, generally by means of glutaraldehydeor periodate. As will be readily recognized, however, a wide variety ofdifferent conjugation techniques exist, which are readily available tothe skilled artisan. Commonly used enzymes include horseradishperoxidase, glucose oxidase, beta-galactosidase and alkalinephosphatase, amongst others. The substrates to be used with the specificenzymes are generally chose for the production, upon hydrolysis by thecorresponding enzyme, of a detectable color change. For example,p-nitrophenyl phosphate is suitable for use with alkaline phosphataseconjugates; for peroxidase conjugates, 1,2-phenylenediamine,5-aminosalicylic acid, or toluldine are commonly used. It is alsopossible to employ fluorogenic substrates, which yield a fluorescentproduct rather than the chromogenic substrates noted above. In allcases, the enzyme-labelled antibody is added to the first antibodyhapten complex, allowed to bind, and then the excess reagent is washedaway. A solution containing the appropriate substrate is then added tothe tertiary complex of antibody-antigen-antibody. The substrate willreact with the enzyme linked to the second antibody, giving aqualitative visual signal, which may be further quantitated, usuallyspectrophotometrically, to give an indication of the amount of haptenwhich was present in the sample. “Reporter molecule” also extends to useof cell agglutination or inhibition of agglutination such as red bloodcells or latex beads, and the like.

Alternately, fluorescent compounds, such as fluorescein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labelled antibody adsorbs the light energy,inducing a state of excitability in the molecule, followed by emissionof the light at a characteristic color visually detectable with a lightmicroscope. As in the EIA, the fluorescent labelled antibody is allowedto bind to the first antibody-hapten complex. After washing off theunbound reagent, the remaining tertiary complex is then exposed to thelight of the appropriate wavelength, the fluorescein observed indicatesthe presence of the hapten of interest. Immunofluorescence and EIAtechniques are both very well established in the art and areparticularly preferred for the present method. However, other reportermolecules, such as radioisotope, chemilluminescent or bioluminescentmolecules, may also be employed. It will be readily apparent to theskilled technician how to vary the procedure to suit the requiredpurpose. It will also be apparent that the foregoing can be used todetect directly or indirectly (i.e., via antibodies) the Lol pIa or LolpIb protein of this invention.

Accordingly, one aspect of the present invention contemplates a methodof detecting Lol pIa or Lol pIb or a derivative or homologue thereof ora allergenic protein immunologically reactive with said Lol pIa or LolpIb or their derivative or homologue in serum, tissue extract, plantextract or other biologically fluid comprising the steps of containingsaid serum, extract or fluid to be tested with an antibody to Lol pIa orLol pIb for a time and under conditions sufficient for an allergenicprotein-antibody complex to form and subjecting said complex to adetecting means. The present invention also contemplates a method ofdetecting an antibody to an allergenic protein from pollen of the familyPoaceae (Gramineae) in serum or other biological fluid comprisingcontacting said serum or fluid with recombinant Lol pIa or Lol pIb ortheir antigenic derivative for a time and under conditions sufficientfor an antibody-Lol pIa or Lol pIb complex to form and subjecting saidcomplex to a detecting means. The latter complex may be detected by theLol pIa or Lol pIb having attached thereto a reporter molecule or byaddition of a second antibody labelled with a reporter molecule.

Accordingly, the present invention is also directed to a kit for therapid and convenient assay for antibodies to Lol pIa or Lol pIb or theirderivatives, homologues or immunological relatives in mammalian bodyfluids (e.g., serum, tissue extracts, tissue fluids), in vitro cellculture supernatants, and cell lysates. The kit is compartmentalized toreceive a first container adapted to an antigenic component thereof, anda second container adapted to contain an antibody to Lol pIa or Lol pIbsaid antibody being labelled with a reporter molecule capable of givinga detectable signal as hereinbefore described. If the reporter moleculeis an enzyme, then a third container adapted to contain a substrate forsaid enzyme is provided. In an exemplified use of the subject kit, asample to be tested is contacted to the contents of the first containerfor a time and under conditions for an antibody, if present, to bind toLol pIa or Lol pIb in said first container. If Lol pIa or Lol pIb of thefirst container has bound to antibodies in the test fluid, theantibodies of the second container will bind to the secondary complex toform a tertiary complex and, since these antibodies are labelled with areporter molecule, when subjected to a detecting means, the tertiarycomplex is detected. Therefore, one aspect of the present invention is akit for the detection of antibodies to a protein having allergenicproperties, said protein from pollen of the family Poaceae (Gramineae),the kit being compartmentalized to receive a first container adapted tocontain recombinant Lol pIa or Lol pIb or their antigenic derivative orhomologue, and a second container adapted to contain and antibody to LolpIa or LoI pIb or their derivative or homologue, said antibody labelledwith a reporter molecule capable of giving a detectable signal. The“report molecule” may also involve agglutination of red blood cells(RBC) on latex beads. In this kit the reporter molecule is aradioisotope, an enzyme, an fluorescent molecule, a chemilluminescentmolecule, bioluminescent molecule or RBC. The kit alternativelycomprises a container adapted to contain recombinant Lol pIa or Lol pIbor their antigenic derivative or homologue labelled with a reportermolecule capable of giving a detectable signal.

Because of the presence of allergens in the environment, hayfever andseasonal asthma continue to have significant morbidity andsocio-economic impact on Western communities, despite advances made intheir pharmacology and immunology. While the available spectrum ofdrugs, including anti-histamines and steroids have resulted inimprovement in the treatment of allergic disease, they have unfortunateside-effects associated with longterm usage. Because of these problems,renewed interest has been shown in the immunotherapy of allergicdisease. Immunotherapy involves the injection of potent allergenextracts to desensitize patents against allergic reactions (Bousquet andMichel (1989) Allergy Clin. Immunol. News 1: 7-10). Unfortunately, thepollen preparations used as allergens are polyvalent and of poorquality. Consequently, concentrations used are frequently high in orderto induce IgG responses, but may be lethal through triggering ofsystemic reactions, including anaphylaxis. The cloned gene product orsynthetic peptides based on the sequence of allergens provides a safermedium for therapy since it can be quality controlled, characterized andstandardized.

The precise mechanism for symptomatic relief remains hypothetical.However, administration of a preparation comprising the protein or atleast one fragment thereof of the instant invention to aryegrass-sensitive individual will modify the allergic response of aryegrass-sensitive individual to ryegrass pollen allergens, e.g., bymodifying the B-cell response to Lol pIa or Lol pIb, the T-cell responseto Lol pIa or Lol pIb, or both responses.

Currently immunotherapy is one of the most frequently administeredtreatments in allergology, and in the USA it is considered the firstchoice. An advantage of this treatment for pollen rhinitis is thattreatment takes up to 3 years, while pharmacotherapy must be carried outduring the patent's entire life time. Patients given pollen extract forimmunotherapy showed a clinical benefit that lasted for four years afterthe end of treatment (Grammer et al. (1984) J. Allergy Clin. Immunol.73: 484-489).

Immune responsiveness to rye-grass pollen allergens Lol pII and Lol pIIIin the human population is significantly associated with thehistocompatibility leukocyte antigen HLA-DR3 (Friedhoff et al.(1988)Tissue Antigens 31: 211-219; Ansari, et al. (1989) Human Immunol. 25:59-71; Ansari et al. (1989) Int Arch. Allergy Appl. Immunol. 88:164-169). This means that the HLA-DR3 encoded class II Ia molecules ofthe antigen-presenting cells may recognize a similar immunodominant Tcell/Ia recognition site present on another allergen. Lol pIa is knownto share an immunodominant T cell/Ia recognition site (YTTEGGTKS EVEDVIP) (SEQ ID NO:26) with both Lol pII and Lol pIII (Friedhoff et al.,supra). Most allergic individuals who respond to Lol pII and III alsorespond to Lol pI, but not the reciprocal. Thus, Lol pIa appears to haveunique T cell/Ia recognition'site(s) not present in Lol pII or III.These unique site(s) appear to be common between Lol pIa and Lol pIb.Certainly, the common T cell/Ia recognition site shared between Lol pIa,II and III is not represented in the deduced sequence of Lol pIb.

Furthermore, it is demonstrated herein that Lol pIa and Lol pIb possessa common B-cell epitope, present in fragment 2P. This common epitope hasbene detected using all three MAbs reactive with Lol pIa. Thisrepresents an epitope that is common between Lol pIa and Lol pIb, butnot present in Lol pII and III, and is likely to be responsible for thedemonstrated concordant responsiveness.

Accordingly, the present invention is directed to Lol pIa and Lol pIb,their derivatives, homologues or immunological relatives includingderivatives containing the common antigenic epitope between Lol pIa andLol pIb which are useful in developing a vaccine to desensitize humansto allergies due to grass pollen.

Accordingly, the present invention contemplates a method fordesensitizing a human allergic to grass pollen which comprisesadministering to said human a desensitizing-effective amount of Lol pIaor Lol pIb, or at least one fragment of Lol pIa or Lol pIb, or aderivative homologue, or immunological relative thereof or combinationsthereof, whether made by recombinant or synthetic means, for a time andunder conditions sufficient to effect desensitization of the human tothe grass pollen.

The present invention, therefore, contemplates a pharmaceuticalcomposition comprising a desensitizing or therapeutically effectiveamount of Lol pIa or Lol pIb, or at least one fragment of Lol pIa or LolpIb or their derivatives, homologues or immunological relatives orcombinations thereof and one or more pharmaceutically acceptablecarriers and/or diluents. The active ingredients of a pharmaceuticalcomposition comprising Lol pIa and/or Lol pIb and/or the like arecontemplated to exhibit excellent therapeutic activity, for example, inthe desensitization of humans allergic to grass pollen when administeredin amount which depends on the particular case. For example, from about0.5 ug to about 20 mg per kilogram of body weight per day may beadministered. Dosage regime may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. The activecompound may be administered in a convenient manner such as by the oral,intravenous (where water soluble), intramuscular, subcutaneous,intranasal, intradermal or suppository routes or implanting (e.g., usingslow release molecules). Depending on the route of administration, theactive ingredients which comprise Lol pIa and/or Lol pIb and/or the likemay be required to be coated in a material to protect said ingredientsfrom the action of enzymes, acids and other natural conditions which mayinactivate said ingredients. For example, the low lipophilicity of LolpIa and/or Lol pIb and/or the like will allow it to be destroyed in thegastrointestinal tract by enzymes capable of cleaving peptide bonds andin the stomach by acid hydrolysis. In order to administer Lol pIa and/orLol pIb and/or the like by other than parenteral administration, theywill be coated by, or administered with, a material to prevent theirinactivation. For example, Lol pIa or the like may be administered in anadjuvant, co-administered with enzyme inhibitors or in liposomes.Adjuvant is used in its broadest sense and includes any immunestimulating compound, such as interferon. Adjuvants contemplated hereininclude resorcinols, non-ionic surfactants such as polyoxyethylene oleylether and n-hexadecyl polyethylene ether. Enzyme inhibitors includepancreatic trypsin. Liposomes include water-in-oil-in-water CGFemulsions as well as conventional liposomes.

The active compounds may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders of the extemporaneous dispersion. In all cases the form must besterile and must be fluid to the extent that easy syringability exists.It must be stable under the conditions of manufacture and storage andmust be preserved against the contaminating action of microorganismssuch as bacteria and fungi. The carrier can be a solvent or dispersionmedium containing for example, water, ethanol, polyol (for example,glycerol, propylene glycol, and liquid polyethylene glycol, and thelike), suitable mixtures thereof, and vegetable oils. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of superfactants. The preventions of theaction of microorganisms can be brought about by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosol, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

When Lol pIa and/or Lol pIb or at least one fragment of Lol pIa and/orLol pIb or the like are suitably protected as described above, theactive compound may be orally administered, for example, with an inertdiliuent of with an assimilable edible carrier, or it may be enclosed inhard or soft shell gelatin capsule, or it maybe compressed into tablets,or it may be incorporated directly with food of the diet. For oraltherapeutic administration, the active compound may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 1% by weightof active compound. The percentage of the compositions and preparationsmay, of course, be carried and may conveniently be between about 5 to80% of the weight of the unit. The amount of active compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained. Preferred compositions or preparations according to thepresent invention are prepared so that an oral dosage unit form containsbetween about 10 ug and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain thefollowing: A binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin may be added or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar or both. A syrup or elixir may contain the active compound,sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound may be incorporated intosustained-release preparations and formulations.

As used herein “pharmaceutically acceptable carrier and/or diluent”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, use thereofin the therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the novel dosageunit forms of the invention are dictated by and directly dependent on(1) the unique characteristics of the active material and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active material for the treatment ofdisease in living subjects having a diseased condition in which bodilyhealth is impaired as herein disclosed in detail.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form as hereinbeforedisclosed. A unit dosage form can, for example, contain the principalactive compound in amounts ranging from 0.5 mg to about 2000 mg.Expressed in proportions, the active compound is generally present infrom about 0.5 mg to about 2000 mg/ml of carrier. In the case ofcompositions containing supplementary active ingredients, the dosagesare determined by reference to the usual dose and manner ofadministration of the said ingredients.

The present invention is further illustrated by the followingnon-limiting Figures and Examples.

EXAMPLES Example 1 Isolation of cDNA Clones

A cDNA expression library in the vector lambda-gt 11 was prepared frompolyadenylated mRNA of mature rye-grass pollen (Beall & Mitchell (1986)J. Immunol. Methods 86: 217-223). This library was screened initiallywith monoclonal antibody (MAb) 40.1 (FIG. 1a).

Poly (A+) mRNA isolated from mature rye grass pollen by the phenolmethod (Herrin and Michaels (1984) Plant Mol. Biol. Reporter 2:24-29)was used to construct a cDNA library in the vector lambda-gt 11. Thelibrary was then screened with antibody probes to detect sequencesexpressing Group I proteins. E. coli Y1090 transfected with 3×10⁴recombinant phages were plated and incubated at 42° C. for 3 h. Theplates were overlaid with a dry 132 mm nitrocellulose (NC) filterspresoaked in 10 mM IPTG and transferred to 37° C. After incubation for 3h the filters were carefully peeled off and incubated in 20 ml perfilter of MTBS (10% w/v non-fat milk powder, 50 mm Tris-HCl, pH 7.6, 150mM NaCl) for 30 min. at room temperature. A second set of NC filters wasplaced on phage plates and after incubating for 3 h were treated asabove. Both sets of NC filters were tested for binding of MAb 40.1 toplaques by the method described in Huynh et al. (1985) in: DNA Cloning,A practical approach, Glover, D. M. (ed.) Vol. 1, pp. 49-78, IRL Press,Oxford, England. The antibody positive plaques were picked, purified,then replated and tested for binding to probes. The positive clones wereplaque-purified and tested for IgE binding using sera from grasspollen-allergic subjects. Eighteen clones were selected as encodingproteins recognized by both Lol pI-specific MAbs and IgE antibodies(Table 1). The largest of the cDNA clones, 1.2 kb in size, thatexpressed rye-grass allergenic protein was initially selected forfurther characterization and sequencing, and designated clone lambda-12R(FIG. 1a).

TABLE 1 Characteristics of cDNA Clones Expressing Group I Allergens ofRye-grass Binding of IgE Approx. Clone Binding Binding from sera Size ofNo. of MAb of MAb of allergic Insert (  R) 12.3^(a) 40.1^(a) idivs. (bp)1 − − − 2 + ++ − 700 3 + ++ − 600 4 + ++ − 800 5 + ++ − 500 6 + ++ − 6007 + ++ − 400 8 − − − 9 − − − 10  − − − 11  + ++ − 500 12  ++ + ++ 1200 (Lol pIb) 13  + ++ + 800 (Lol pIa) 14  ++ + + 1200  15  − − − 16  + ++ −800 17  + ++ − 400 18  ++ + + 1200  ++ : -strongest binding − : -nobinding MAb 12.3 shows high affinity for Lol pIb (clone 12R). MAb 40.1shows high affinity for Lol pIa (clone 13R).

The specificity of IgE and MAbs was tested by immunoblot analysis ofrye-grass pollen protein extracts (FIG. 1b).

Soluble proteins were extracted from rye-grass pollen by vigorousshaking in PBS (150 mM, pH 7.2) on ice for 3 h. Pollen was spun out ofsolution and the extracted protein standardized using the Biorad assay.120 ug protein per lane was electrophoresed under reducing conditions ona 10-15% w/v SDS-polyacrylamide gel. Proteins were electroblotted ontoNC filters and the blot blocked with TBS (10 NiM Tris, 150 mM NaCl, pH7.9) containing 10% w/v non-fat milk powder. The blot was cut intostrips and each treated with the various probes: MAbs were diluted1:1000 in TBS containing 1% BSA. Sera collected from at least 4 patientswith high RAST scores for grass pollen, was pooled and used diluted 1:5in TBS/1% w/v BSA for IgE binding. Horseradish peroxidase-conjugatedsecondary antibodies were used (Dakopatts) and after washing, bindingwas visualized with 4-chloro 1-naphthol (Biorad) and H₂O₂.

When the immunoblot was incubated in pooled sera from grasspollen-allergic individuals, strong IgE binding was observed throughoutthe 28-35 kD region. The MAbs used in this study, 3.2, 12.3, 21.3 and40.1 had previously been partially characterized (Kahn and Marsh (1986)Molec. Immunol. 23: 1281-1288; Singh and Knox (1985) Intl. Arch..Allergy and Applied. Immunol. 78: 300-304; Smart et al. (1983) Intl.Arch. Allergy and Applied Immunol. 72: 243-248). MAbs 3.2, 21.3 and 40.1showed strong reactivity with the proteins in the 28-35 kD region. MAb12.3 exhibited no binding to the 35 Kd band, but bound strongly to thelower bands. These interactions suggest that both IgE and MAbs canrecognize denatured allergens, which makes them suitable probes for thedetection of recombinant protein express in E. coli.

The allergen-beta-galactosidase fusion protein produced by the inductionof lysogenic cultures of lambda-12R, was characterized by immunoblotanalysis using MAb 40.1. This fusion protein of approximately 146 kD isassumed to be comprised of the 116 kD beta-galactosidase and 30 kD ofallergen-encoded sequence. This fusion protein was produced in lowyields. So in order to increase yields of the cloned allergen forfurther analysis, we used an alternative expression system. The 1.2 kbinsert was subcloned in the pGEX1-3 series of plasmid expressionvectors. These plasmids give a fusion polypeptide with the carboxylterminus of the Schistosoma Japonicum glutathione S-transferase protein(Smith and Johnson (1988) Gene 67: 31-40). Strong IgE binding wasdetected only in bacteria transformed with pGEX-12R, and not in thosewith parental pGEX plasmids (data not shown, but similar binding shownin FIG. 4). Probing of Western blots with control sera that had negativeradioallergosorbent (RAST) score for rye-grass pollen showed no IgEbinding.

Example 2 Identity of Cloned Allergen 12R and 13R

All four MAbs used in this study recognized the cloned allergen 12R(FIG. 1a).

Not all MAbs show the same specificity to the native Lol pI proteins(FIG. 1b). In particular, MAb 12.3 does not recognize the 35 kD band.Because the cloned allergen binds all the MAbs, and with high intensityto MAb 12.3, it is predicted that the cloned allergen is likely tocorrespond to a protein of lower Mr, and not to the 35 kD protein. Toconfirm its identity, an immunological approach developed for parasiteantigens was employed (eg Beall & Mitchell (1986) J. Immunol. Methods86: 217-223). In this method, the cloned allergen 12R was immobilized onnitrocellulose membrane, and used to bind specific IgE antibodies fromsera. Bound antibodies were eluted and used to probe a Western blot ofrye-grass pollen proteins. Highly specific and reproducible patterns ofbinding were consistently obtained in several experiments to two proteincomponents of molecular weight 31 and 33 kD. No specific binding wasobserved when IgE antibodies from non-grass pollen allergic individualswere used now when extracts of E. coli transformed with non-recombinantpGEX plasmids were used to select IgE antibodies.

These experiments demonstrate that IgE antibodies that bind to clone 12Rrecognize two components with slightly different molecular weights, 31and 33 kD. The 31/33 and 35 kD components may be structurally differentin terms of their physico-chemical characteristics, and are tentativelydesignated Lol pIa (clone 13, 35 kD component) and Lol pIb (31/33 kdcomponents)

To test this hypothesis, Lol pIa and Lol pIb proteins were purified bytwo-dimensional analysis involving preparative iso-electric focusing inthe first dimension, followed by SDS-PAGE of the individual fractionscollected. This procedure successfully separated Lol pIa (pI 5.5) andLol pIb (pI 9.0) in sufficient quantity for their N-terminal sequence tobe determined (Table 2).

TABLE 2 N-Terminal Amino Acid Sequences of Grass Pollen AllergensObtained In This Study Compared with Reported Sequences AllergenN-terminal sequence Lol pI IAKV?PG??I TAEYGDKWLD AKSTWYGKPT Lol pIaIAKVPP*GP*WI TAEYGDKWLD AK?T------ Clone 13R IAKVPPGPPNI TAEYGDKWLDAKSTWYGKPT Lol pIb ADAGYTPAA? ?TPATPA?T Clone 12R ADAGYTPAAA ATPATPAATPAGGWRE Lol pII AAPVEFTVEK GSDEKNLALS IKYNKEGDSMA Lol pIII -TKVDLTVEKGSDAKTLVLN IKYTRPGDTLA *Indicates Hydroxyproline residue. The N-terminalsequences in Table 2 have the following Sequence Listing numbers:LolpI-SEQ ID NO: 19; Lol pIa-SEQ ID NO: 20; Clone 13R-SEQ ID NO: 21; LolpIb-SEQ ID NO: 22; Clone 12R - SEQ ID NO: 23; Lol pII-SEQ ID NO: 24, LolpIII-SEQ ID NO: 25.

Individual protein components were isolated using preparativeisoelectric focussing on the Rotofor (Biorad). The proteins wereseparated on SDS-PAGE, and transferred to PVDF membrane (Millipore).N-terminal sequencing was performed according to Matsudaira (1987) J.Biol. Chem. 262: 10035-10038, and Simpson et al. (1989) J. Chromatogr.476: 345-361.

The sequence of the 35 kD allergen shows homology with the previouslypublished sequence of Lol pI (Table 2). The 31/33 kD protein, Lol pIb,has a different N-terminal amino acid sequence from Lol pIa. It isconcluded that the allergen encoded by clone 12R represents a majornewly identified allergen, Lol pIb and that clone 13R encodes allergenLol pIa. The nucleotide sequences of clones 12R (SEQ ID NO: 1) and 13R(SEQ ID NO: 3) are shown in FIGS. 3 and 6, respectively. The predictedamino acid sequences are also shown in SEQ ID NO: 2 (clone 12R) and SEQID NO: 4 (clone 13R).

Clones 4R, 6R, 16R and 17R (Table 1) were also sequenced and found to bepartial clones of Lol pIa (SEQ ID, NO: 5). The relative position of thesequenced clones with respect to the full-length nucleotide sequence ofLol pIa ID NO: 5) is shown in Table 3.

TABLE 3 Summary of antibody binding to Lol pI cDNA clones NucleotidePosition in Lol pIa Clone FMC-A1 FMC-A7 IgE sequence  4R ++ + −  0-764 6R ++ + − 159-754 16R ++ + −  12-764 17R ++ + − 383-756

Example 3 Pollen-Specific Expression of Allergens

Poly A+ RNAs were isolated from different plant tissues: seed, leaf,root and pollen. 20 ug of total RNA from the different tissues waselectrophoresed on a 1.2% w/v agarose gel in the presence of formamideand formaldehyde (Sambrook et al., supra), transferred to Hybond-C extra(Amersham, Arlington Heights, Ill.) and the filters baked at 80° C. for2 h. The 1.2 kb 12R cDNA was radio-labelled with ³²P and incubated withthe NC filter at 65° C. in the presence of 50% v/v formamide. Themembrane was washed with 2×SSC containing 0.1% w/v SDS at 65° C.

Proteins were isolated from the different tissues (flower, leaf, rootand pollen) by grinding in 10 mM PBS containing 1 mM PMSF, andimmunoblotted (10 ug protein per lane) with the indicated antibodies.The binding was visualized by using ¹²⁵I-goat anti-mouse Ig (Amersham)for MAbs, and polyclonal ¹²⁵I-goat anti-human IgE (Kallestad, USAfollowed by autoradiography.

Northern blot analysis of RNA prepared from pollen showed high levels ofexpression of the cloned allergen gene in pollen but not in anyvegetative tissues. A prominent band approximately 1.3 kb long is notdetectable in RNA from vegetative tissues (FIG. 2a). Pollen-specific RNAexpression corresponded to pollen-specific expression of antigensrecognized by MAbs 40.1, 12.3 and IgE antibodies (FIG. 2b). Specificbinding occurred only when pollen and floral tissues (containing pollen)were used as protein source.

Example 4 Primary Structure Analysis

The cDNA clone 12R was isolated, and subcloned into pGEM-3Z vectors(Promega, Madison, Wis.), restriction mapped, and resubcloned in varioussized restriction fragments into pGEM vectors. DNA sequence wasdetermined by double-stranded sequencing carried out by the dideoxychain termination method (Sanger et al. (1977) Proc. Natl Acad. Sci. USA74: 5463-5468), using Sequenase (US Biochemical) and T7 DNA polymerase(Pharmacia, Piscataway, N.J.). Sequencing was carried out concurrentlywith both ddNTPs and 7-deaza dGTP. The reading frame was confirmed bysequencing two expression subclones in pGEX vector as detailed in FIG.4. DNA sequence data were analyzed using the MELBDBSYS system (NBRFProtein Identification Resource, Washington, USA; GENBANK, Los AlamosNational Laboratory, USA; EMBL, Heidelberg, Germany; Swissprot and theNBRF PIR protein databases).

The nucleotide sequence of the cDNA clone 12R (SEQ ID NO: 1) is GC-rich(68% GC, FIG. 3b). There is an open reading frame of 921 bp startingwith an ATG initiation codon at position 40 and terminating with a TGAcodon at position 964. The proposed translation initiation site and itsflanking sequences share 89% homology with the consensus plant sequenceAACAATGGC (positions 36-44), and can be considered as in optimum contextwith the presence of a purine at position −3 from the methionine codon.The open reading frame potentially encodes a protein of Mr 34.1 kD.

The predicted protein sequence, which is rich in alanine (23%) andproline (13%), has a putative signal or target peptide sequence of 25amino acids (FIG. 3b) (SEQ ID NO: 2). This is indicative of a cleavedprotein of Mr 31.3 kD. The N-terminal protein sequence of Lol pIb isidentical to the deduced amino acid sequence of clone 12R immediatelyafter the putative cleavage site of the signal peptide sequence. Thisconfirms that the cDNA-12R encodes the Lol pIb allergenic protein andthat the protein has a signal peptide sequence which is cleaved.

The signal sequence has features that are typical of other eukaryoticsequences: a relatively hydrophilic sequence of 5 amino acids at theC-terminus, a relatively hydrophobic sequence extending over most of thesignal region which becomes more hydrophilic at the N-terminus (FIG.3c). The amino acids at the C-terminus include alanine at the cleavagesite, an aromatic residue tyrosine at −2, and a helix breaker proline at−6, all of which are common features of the C-terminal region of signalsequence.

A search of existing data-bases indicates no homology between thededuced amino acid sequence of lambda-12R and any other known protein.Furthermore, a search for consensus glycosylation sequences(Asn-x-Ser/Thr) in the deduced amino acid sequence detected no suchsequences. The absence of an N-linked carbohydrate chain on the allergenwas confirmed by the lack of deglycosylation following treatment withthe enzymes N-glycanase and endo-F glycosidase. Chemical deglycosylationfollowed by SDS-PAGE showed no decrease in molecular weight of theprotein. The 31/33 kD components remained as a doublet, suggesting thatthe difference in molecular weight is not due to glycosylation. Thedeglycosylation treatments did not affect IgE binding to the 31/33 kDcomponents. As compared to Lol pIa which has 5% carbohydrate, nocarbohydrate is present in Lol pIb.

Example 5 Delineation of IgE- and MAb- Reacting Epitopes

To localize MAb and IgE determinants, an E. coli recombinant expressionsystem was employed (Smith and Johnson (1988) Gene 67: 31-40). Usingthis system, a number of restriction fragments were subcloned into theexpression plasmid PGEX 1-3. The “in frame” sub-cloning of full lengthcDNA into PGEX, expressed the 61 kD fusion protein recognized by bothIgE and MABs 40.1 and 12.3.

The full length cDNA 12R (SEQ ID NO: 1) or two restriction fragments 1Hand 2P, were subcloned into plasmid expression vector pGEX. Theprocedure for inducing fusion proteins and preparation of bacteriallysates have been described earlier (Smith and Johnson, supra). Thelysates obtained were subjected to reducing SDS-PAGE, followed bytransfer to NC membranes. The blots were probed with IgE antibodies, andMAbs 40.1, 12.3 as described in FIG. 1b, except that ¹²⁵I-anti-human IgE(Kallestad) was used to detect IgE binding.

Immunoblot analysis showed that most of the fusion protein produced iscleaved by bacterial proteases near its fusion site with glutathione-Stransferase, generating break-down products which are recognized by IgEantibodies (FIG. 4). The recombinant fusion protein expressed byfragment 2P, although strongly reactive with both MAbs, was notrecognized by IgE antibodies in pooled allergic sera. However, theN-terminally truncated protein produced by fragment 1H was notrecognized by either of the MAbs, but was highly reactive with the IgEantibodies.

In this way, two distinct domains of the allergen molecule have beendelineated: the N-terminal containing fragment has recognition sites forMAbs 12.3 and 40.1; and the C-terminal containing fragment 1H whichshows strong IgE binding and thus has the allergenic determinant(s).Because the two MAbs have different binding specifities (FIG. 1b), therecognition sites for the two MAbs are likely to be different, althoughin the same fragment. Fine mapping with smaller fragments is needed todelineate the 12.3 and 40.1 binding sites, but these results aresufficient to show that the IgE determinant is different.

Example 6 Intracellular Targeting of Lol pIb in Rye-Grass Pollen

Mature pollen of Lolium perenne was prepared for scanning electronmicroscopy according to established methods (Staff et al. (1990)Histochem J. 22: 276-290). For immunocytochemistry, mature anthers werefixed under anhydrous conditions: 0.1% glutaraldehyde, 1%paraformaldehyde in 2,2-dimethoxypropane at 4° C. for 2 h and processedfor transmission electron microscopy (Staff et al., supra). This methodhas been developed to reduce diffusion of the allergens from theircellular sites in aqueous media. Blocks were polymerized in LR goldresin with 1% benzil at −25° C. under UV illumination and 80 nm thinsections picked up on gold grids. Immuno-labelling was first withprimary antibody, MAb 12.3 (specific for Lol pIb) followed bygold-goat-anti-mouse IgG probe (15 nm particle size). This label wassilver-enhanced to 40 nm particle size (modified from Danscher &Norgaard (1983) J. Histochem. Cytochem. 31:1394-1398. A second labellingwas performed on the same sections with a mixture of three MAbs, 3.2,21.3 and 40.1 (specific for Lol pIa) followed by gold-goat-anti-mouseIgG probe with 15 nm particle size. Antibody specificity and methodcontrols run as described previously (Staff et al., supra) showed nogold particles at these sites.

Lol pI is located in the cytosol and not in the organelles (Staff etal., supra). These findings were obtained using immuno-gold probes withMAbs specific for Lol pI. As shown herein, MAb 12.3, which is specificfor pIb, binds predominantly to the starch grains (FIG. 5a, b). Grasspollen is filled with starch grains which are 1×2.5 um in size, andoriginate in the lumen of amyloplasts.

As shown in FIG. 5b, the large gold particles located predominantly overthe starch grains (large electron-lucent spaces) show binding of MAb12.3 to Lol pIb, while smaller particles over the cytosol are typical ofbinding to Lol pIa. Scale bar is 1 um. FIG. 5c shows the appearance offresh, viable pollen after exposure to water for 30 s, dark fieldillumination. Most pollen grains burst, extruding their cytoplasmiccontents, including starch grains (white particles) through the germinalpore. Scale bar, 30 um.

The localization of Lol pIb in the plastids implies that this proteinshould be transported from the cytosol to the lumen of the plastidsduring development. For transport to chloroplasts, the proteins whichare synthesized in the cytosol are synthesized as large precursorscontaining a target peptide sequence that is cleaved after transportinto the organelle. Comparison of the signal sequence of Lol pIb (FIG.3b) (SEQ ID NO: 2, amino acids −25 through −1) with the domain structureof published mitochondrial and chloroplast-specific transit peptides isas below.

For import into plastids, plant signal peptides need additionalinformation at the carboxyl terminus, which resides in −2 to −7 regionfrom the cleavage site of the peptide (SEQ ID NO: 2). The signal peptideof most chloroplast-targeted proteins possesses the sequence “G-R-V” orfunctionally homologous sequence reading from the −2 position. Thesignal peptide of Lol pIb (clone 12R) has the sequence “G-R-S” in thisposition (FIG. 3b). Thus it is concluded that the Lol pIb molecule issynthesized first as a pre-allergen in the cytosol, and is transportedto the plastid for post-translational modification. These intracellularprocessing steps may explain the appearance of the doublet 31/33 kDfound by immunoblotting. The unprocessed pre-allergen is 33 kD, andafter processing in the plastids, the mature protein is 31 kD. Boththese forms co-exist in mature pollen. This doublet may also representdifferent isoforms of Lol pIb.

Example 7 Presentation of Lol pIa and b to the Immune System

When the rye-grass flower opens, the anthers are exerted and the pollenis released into the air through a pore which opens at the base of eachanther. Rye-grass shows the greatest pollen production of any grass,releasing approximately 460 kg of pollen per hectare into the atmospherein pastures that are not mowed or grazed. Ninety-nine per cent of thispollen is deposited (and re-deposited) within 1 km of its source. Grasspollen is short-lived, yet it can remain for several days in theatmosphere. Experiments show that the pollen remains viable for only afew hours after release.

When viable, the grains can germinate on the stigma, or in artificialmedia with high levels of osmoticum. Living viable rye-grass pollengrains when exposed to water, burst at the single germinal aperturereleasing the cytoplasmic contents (FIG. 5c). Prominent among thereleased contents are the starch grains. Media with high osmoticum, e.g.30% w/v sucrose are required to maintain tonicity of the grains. Incontrast, it is well-known that dead pollen grains which have nopermeability barriers, act like a sponge. Cellular proteins, includingallergens, are released from the surface upon moistening.

It is easy to see how grass pollen can trigger hay fever aftercontacting the oral and eye mucosa, by direct release of the allergens.The pollen grains themselves remain on the surface of the mucosa, butthe released allergenic proteins pass through the mucosa andsubepithelial layers where they interact with basophils and mast cells.It is less easy to see how pollen grains as large as 30-50 um indiameter can induce allergic asthma, a disease triggered by the presenceof allergens in the airways of the lungs.

Recent evidence suggests that grass pollen allergens are associated withsmaller micronic particles found in the atmospheric aerosol. Theoriginal of such particles is obscure. From the present results onallergen localization, and observations on pollen behavior in water, anew hypothesis is proposed to explain how grass pollen can induceallergic asthma in the lungs of susceptible humans. Starch grains arereleased as micronic particles into the atmospheric aerosol when theliving pollen grains encounter water vapor, or water on the surface of aleaf or other substrata. These particles, both coated and filled withallergens, act as vehicles for allergen presentation to the upper andlower respiratory tract. Micronic particles can also, of course, resultsfrom the leaching of allergens from grass pollen and deposition on othercomponents of the atmospheric aerosol.

Example 8 Isolation and Cloning of Nucleic Acid Sequence Coding for LolpIa

Total mRNA was extracted from mature ryegrass pollen by the phenolmethod of Herrin and Michaels,supra. Double-stranded cDNA wassynthesized from 1 μg of total mRNA using a commercially available kit(cDNA synthesis system plus kit, BRL, Gaithersburg, Md.). After a phenolextraction and ethanol precipitation, the cDNA was blunted with T4 DNApolymerase (Promega, Madison, Wis.), and ligated toethanol-precipitated, self-annealed AT (SEQ ID No.: 7) and AL (SEQ IDNO: 8) oligonucleotides for use in a modified Anchored PCR reaction,according to the method in Rafnar et al. (1991) J. Biol. Chem. 266:1229-1236; Frohman et al. (1990) Proc. Natl. Acad. Sci. USA 85;8998-9002: and, Roux et al. (1990) BioTech. 8: 48-57. Oligonucleotide AThas the sequence 5′-GGGTCTAGAGGTACCGTCCGATCGATCATT-3′ (SEQ ID NO: 7)(Rafnar et al. supra). Oligonucleotide AL has the sequence AATGATCGATGCT(SEQ ID NO: 8) (Rafnar et al. supra.).

Polymerase chain reactions (PCR) were carried out using a commerciallyavailable kit (GeneAmp® DNA Amplification kit, Perkin Elmer Cetus,Norwalk, Conn.) whereby 10 μl 10× buffer containing dNTPs was mixed with1 μg each of primer AP, which has the sequence5′-GGGTCTAGAGGTACCGTCCG-3′ (SEQ ID NO: 9) (Rafner et al. supra.) andLpA-5, which has the sequence 5′-CCCTGCAGATTATTTGAGATCTTGAG-3′ (SEQ IDNO: 10), cDNA (3-5 μl of a 20 μl Tinkered cDNA reaction mix), 0.5 μlAmplitaq DNA polymerase, and distilled water to 100 μl.

Nucleotides 1 through 8 (5′-CCCTGCAG) of LpA-5 (SEQ ID NO: 10)correspond to a Pst I site added for cloning purposes; the remainingnucleotides correspond to the non-coding strand sequence complementaryto nucleotides 483 through 500 of SEQ ID NO: 3.

The samples were amplified with a programmable thermal controller (MJResearch, Inc., Cambridge, Mass.). The first 5 rounds of amplificationconsisted of denaturation at 94° C. for 1 minute, annealing of primer tothe template at 45° C. for 1.5 minutes, and chain elongation at 70° C.for 2 minutes. The final 20 rounds of amplification consisted ofdenaturation as above, annealing at 55° C. for 1.5 minutes, andelongation as above. Five percent (5 μl) of this initial amplificationwas then used in a secondary amplification whereby 10 μl 10× buffercontaining dNTPs was mixed with 1 μg each of primer AP (SEQ ID NO: 9)and primer LpA-3, which has the sequence5′-CCCTGCAGTCATGCTCACTTGGCCGAGTA-3′ (SEQ ID NO: 11), 0.5 μl Amplitaq DNApolymerase, and distilled water to 100 μl. The secondary PCR reactionwas performed as described herein. Nucleotides 1 through 8(5′-CCCTGCAG-3′) of LpA-3 (SEQ ID NO: 11) correspond to a Pst I siteadded for cloning purposes; nucleotides 9 through 12 (5′-TCA-3′)correspond to the complementary sequence for a new stop codon, and theremaining nucleotides correspond to the non-coding strand sequencecomplementary to nucleotides 793 through 810 of SEQ ID NO: 5(nucleotides 426 through 443 of SEQ ID NO: 3), including translatedsequence of Lol pIa (SEQ ID NO: 5), the native stop codon and 3′untranslated sequence.

Amplified DNA was recovered by sequential chloroform, phenol, andchloroform extractions, followed by precipitation at −20° C. with 0.5volumes of 7.5 ammonium acetate and 1.5 volumes of isopropanol. Afterprecipitation and washing with 70% ethanol, the DNA was simultaneouslydigested with Xba I and Pst I in a 15 μl reaction and electrophoresedthrough a preparative 3% GTG NuSieve low melt gel (FMC, Rockport, Me.).The appropriate sized DNA band was visualized by EtBr staining, excised,and ligated into appropriately digested M13mp18 for sequencing by thedideoxy chain termination method (Sanger et al. (1977) Proc. Natl AcadSci USA 74: 5463-5476) using a commercially available sequencing kit(Sequenase kit, U.S. Biochemicals, Cleveland, Ohio).

Both strands were sequenced using M13 forward and reverse primers (N.E.BioLabs, Beverly, Mass.) and internal sequencing primers LpA-13 (SEQ IDNO: 12), LpA-12 (SEQ ID NO: 13), LpA-9 (SEQ ID NO: 14), LpA-2 (SEQ IDNO: 15), LpA-7 (SEQ ID NO: 16), LpA-10 (SEQ ID NO: 17), and LpA-IA (SEQID NO.: 18). LpA-13 has the sequence 5′-GAGTACGGCGACAAGTGGC-3′ (SEQ IDNO: 12), which corresponds to nucleotides 121 through 139 of SEQ ID NO:5. LpA-12 has the sequence 5′-TTCGAGATCAAGTGCACC-3′ (SEQ ID NO: 13),which corresponds to nucleotides 310 through 318 of SEQ ID NO: 5. LpA-9has the sequence 5′-GTGACAGCCTCGCCGG-3′ (SEQ ID NO: 14), whichcorresponds to the non-coding strand sequence complementary tonucleotides 335 through 350 of SEQ ID NO: 5. LpA-2 has the sequence5′-GGGAATTCCATGGCGAAGAAGGGC-3′ (SEQ ID NO: 15). Nucleotides 1 through 7(5-GGGATT-3′) of SEQ ID NO: 15 correspond to part of an Eco-RIrestriction site added for cloning purposes; the remaining sequence ofSEQ ID NO: 15 corresponds to nucleotides 425 through 441 of SEQ ID NO:5. LpA-7 has the sequence 5′-GTGCCGTCCGGGTACT-3′ (SEQ ID NO: 16), andcorresponds to non-coding strand sequence complementary to nucleotides503 through 518 of SEQ ID NO: 5. LpA-10 has the sequence5′-CCGTCGACGTACTTCA-3′ (SEQ ID NO: 17), which corresponds to non-codingstrand sequence complementary to nucleotides 575 through 590 of SEQ IDNO: 5. LpA-IA has the sequence 5′-GGAGTCGTGGGGAGCAGTC-3′ (SEQ ID NO:18), which corresponds to nucleotides 654 through 672 of SEQ ID NO: 5.

Multiple clones from several independent PCR reactions were sequenced.The sequence of a representative clone of Lol pIa, clone 26.j, with thededuced amino acid sequence is shown in FIGS. 7a and 7b (SEQ ID NO: 5).As shown in FIGS. 7a and 7b, the nucleic acid sequence coding for LolpIa has an open reading frame beginning with an ATG initiation codon atnucleotide 16 (SEQ ID NO: 5, nucleotide base 16) and ending with a TGAstop codon at nucleotide 805 (SEQ ID NO: 5, nucleotide base 805). Thetranslated protein has a deduced amino acid sequence of 263 amino acidswith a predicted molecular weight and pI of 28.4 kD and 5.55respectively. The initiating methionine is numbered amino acid −23, withamino acid numbered +1 corresponding to the NH2-terminus of the matureprotein, as defined by amino acid sequencing (Cottam et al (1986)Biochem. J. 234: 305-310). Amino acids −23 through −1 in FIG. 7 (SEQ IDNO: 5, amino acids −23 through −1) correspond to a leader sequence thatis cleaved from the mature protein; the mature protein is thereforecomposed of 240 amino acids and has a predicted molecular weight and pIof 26.1 kD and 5.38 respectively. There is a single potential N-linkedglycosylation site at amino acid 9.

Amino acids 1 through 30 of clone 26.J (SEQ ID NO: 5) correspond exactlyto the published sequence of the NH₂ terminus of Lol pI (Cottam et al.,supra). Amino acids 213 through 240 of clone 26.j (SEQ ID NO: 5)correspond exactly to the published internal amino acid sequence of Lp;pI (Esch and Klapper (1989) Mol. Immunol. 26: 557-561).

The first nucleotide of clone 13R (SEQ ID NO: 3) corresponds tonucleotide 368 of Lol pIa (SEQ ID NO: 5).

Example 9 Identification of Polymorphisms in Lol pIa

A number of polymorphisms in the nucleotide sequence coding for Lol pIawere discovered during the amplification and sequencing of different LolpIa clones. Some of the polymorphisms cause an amino acid changerelative to that of clone 26.j, while others are silent polymorphismsthat do not cause an amino acid change. The polymorphisms found in thesequence coding for Lol pIa are summarized in Table 4. The nucleotidebase numbers are those of the sequence of clone 26.j shown in FIGS. 7aand 7 b (SEQ ID NO: 5).

TABLE 4 POLYMORPHISMS DETECTED IN Lol pIa Nucleotide Polymorphism AminoAcid Polymorphism 1 GGC₂₁₅→GGA/GGT NONE 2 G₂₃₄AC₂₃₆→GAT D₄₅→N 3GTT₂₃₉→GTC NONE 4 CGT₃₅₁→CGC NONE 5 GGC₃₅₆→GGT NONE 6 AAC₃₈₉→AAT NONE 7CCC₃₉₈→CCT NONE 8 CAT₄₁₃→CAC NONE 9 GCC₄₃₄→GCA NONE 10 GAC₅₃₀→GAT NONE11 GG₅₃₂C→GAC G₁₄₄→D 12 CCG₅₄₂→CCA NONE 13 ACA₅₄₅→ACG NONE 14 GC₅₆₂T→GGTA₁₅₄→G 15 CTC₅₈₁→CTG NONE 16 GCG₆₂₆→GCC NONE 17 ATC₇₈₂→ATT NONE 18CCT₇₈₅→CCC NONE

All confirmed nucleotide polymorphisms (polymorphisms observed in thesequence analysis of clones from two independent PCR reactions) areshown relative to the sequence of claim 26.j (SEQ ID NO: 5). Thepolymorphic residues in their respective codon triplets are numbered.Productive amino acid changes are also shown; most nucleotidepolymorphisms are silent and do not result in an amino acid change.Twenty-eight potential polymorphisms have only been observed in clonesfrom single PCR reactions. Seventeen of these 28 potential polymorphismsare silent mutations and do not result in an amino acid polymorphism;the remaining 11 potential polymorphic sites would result in thefollowing amino acid changes, specifically, T₁₁→M, A₄₉→V, R₆₇→S,K_(79→R, V) ₉₀→I, Q₁₃₃→R, I₁₈₂→T, V₁₇₃→E, I₁₈₇→T, V₂₂₃→F and K₂₃₂→R.

Those skilled in the art will appreciate that the invention described issusceptible to variations and modification other than those specificallydescribed. It is understood that the invention includes all suchvariations and modifications. The invention also includes all steps,features, compositions and compounds referred to or indicated in thisspecification, individually or collectively, and any and allcombinations of any two or more of said steps or features.

The nucleotide sequences presented herein represent the most accuratedata presently available. Minor corrections may subsequently be made tothe sequences without departing from the scope of the present invention.

26 1242 base pairs nucleic acid single linear cDNA not provided CDS40..963 mat_peptide 115..963 1 CGCTATCCCT CCCTCGTACA AACAAACGCAAGAGCAGCA ATG GCC GTC CAG AAG 54 Met Ala Val Gln Lys -25 TAC ACG GTG GCTCTA TTC CTC CGC CGT GGC CCT CGT GGC GGG CCC GGC 102 Tyr Thr Val Ala LeuPhe Leu Arg Arg Gly Pro Arg Gly Gly Pro Gly -20 -15 -10 -5 CGC TCC TACGCC GCT GAC GCC GGC TAC ACC CCC GCA GCC GCG GCC ACC 150 Arg Ser Tyr AlaAla Asp Ala Gly Tyr Thr Pro Ala Ala Ala Ala Thr 1 5 10 CCG GCT ACT CCTGCT GCC ACC CCG GCT GGC GGC TGG AGG GAA GGC GAC 198 Pro Ala Thr Pro AlaAla Thr Pro Ala Gly Gly Trp Arg Glu Gly Asp 15 20 25 GAC CGA CGA GCA GAAGCT GCT GGA GGA CGT CAA CGC CTG GCT TCA AGG 246 Asp Arg Arg Ala Glu AlaAla Gly Gly Arg Gln Arg Leu Ala Ser Arg 30 35 40 CAG CCG TGG CCG CCG CTGCCA ACG CCC CTC CGG CGG ACA AGT TCA AGA 294 Gln Pro Trp Pro Pro Leu ProThr Pro Leu Arg Arg Thr Ser Ser Arg 45 50 55 60 TCT TCG AGG CCG CCT TCTCCG AGT CCT CCA AGG GCC TCC TCG CCC ACC 342 Ser Ser Arg Pro Pro Ser ProSer Pro Pro Arg Ala Ser Ser Pro Thr 65 70 75 TCC GCC GCC AAG GCA CCC GGCCTC ATC CCC AAG CTC GAC ACC GCC TAC 390 Ser Ala Ala Lys Ala Pro Gly LeuIle Pro Lys Leu Asp Thr Ala Tyr 80 85 90 GAC GTC GCC TAC AAG GCC GCC GAGGCC CAC CCC CGA GGC CAA GTA CGA 438 Asp Val Ala Tyr Lys Ala Ala Glu AlaHis Pro Arg Gly Gln Val Arg 95 100 105 CGC CTT CGT CAC TGC CCT CAC CGAAGC CTC CGC GTC ATC GCC GGC GCC 486 Arg Leu Arg His Cys Pro His Arg SerLeu Arg Val Ile Ala Gly Ala 110 115 120 CTC GAG GTC CAC GCC GTC AAG CCCGCC ACC GAG GAG GTC CTC GCT GCT 534 Leu Glu Val His Ala Val Lys Pro AlaThr Glu Glu Val Leu Ala Ala 125 130 135 140 AAG ATC CCC ACC GGT GAG CTGCAG ATC GTT GAC AAG ATC GAT GCT GCC 582 Lys Ile Pro Thr Gly Glu Leu GlnIle Val Asp Lys Ile Asp Ala Ala 145 150 155 TTC AAG ATC GCA GCC ACC GCCGCC AAC GCC GCC CCC ACC AAC GAT AAG 630 Phe Lys Ile Ala Ala Thr Ala AlaAsn Ala Ala Pro Thr Asn Asp Lys 160 165 170 TTC ACC GTC TTC GAG AGT GCCTTC AAC AAG GCC CTC AAT GAG TGC ACG 678 Phe Thr Val Phe Glu Ser Ala PheAsn Lys Ala Leu Asn Glu Cys Thr 175 180 185 GGC GGC GCT ATG AGA CCT ACAAGT TCA TCC CCT CCC TCG AGG CCG CGG 726 Gly Gly Ala Met Arg Pro Thr SerSer Ser Pro Pro Ser Arg Pro Arg 190 195 200 TCA AGC AGG CCT ACG CCG CCACCG TCG CCC GCC GCG CCC GAG GTC AAG 774 Ser Ser Arg Pro Thr Pro Pro ProSer Pro Ala Ala Pro Glu Val Lys 205 210 215 220 TAC GCC GTC TTT GAG GCCGCG CTG ACC AAG GCC ATC ACC GCC ATG ACC 822 Tyr Ala Val Phe Glu Ala AlaLeu Thr Lys Ala Ile Thr Ala Met Thr 225 230 235 CAG GCA CAG AAG GCC GGCAAA CCC GCT GCC GCC GCT GCC ACA GCG GCC 870 Gln Ala Gln Lys Ala Gly LysPro Ala Ala Ala Ala Ala Thr Ala Ala 240 245 250 GCA ACC GTT GCC ACC GCGGCC GCA ACC GCC GCC GCC GTG CTG CCA CCG 918 Ala Thr Val Ala Thr Ala AlaAla Thr Ala Ala Ala Val Leu Pro Pro 255 260 265 CCG CTG CTG GTC GTA CAAAGC CTG ATC AGC TTG CTA ATA TAC TAC 963 Pro Leu Leu Val Val Gln Ser LeuIle Ser Leu Leu Ile Tyr Tyr 270 275 280 TGAACGTATG TAAGTGCATG ATCCGGGCGGCGAGTGGTTT TGTTGATAAT TAATCTTCGT 1023 TTTCGTTTTC ATGCAGCCGC GATCGAGAGGTTGCATGCTT GTAATAATTC AATATTTTTC 1083 ATTTCTTTTT GAATCTGTAA ATCCCCATGACAAGTAGTGG GATCAAGTCG GCATGTATCA 1143 CCGTTGATGC GAGTTTAACG ATGGGGAGTTTATCAAAGAA TTTATTATTA AAAAAAAAAA 1203 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAAAAAAAAAAA 1242 308 amino acids amino acid linear protein not provided 2Met Ala Val Gln Lys Tyr Thr Val Ala Leu Phe Leu Arg Arg Gly Pro -25 -20-15 -10 Arg Gly Gly Pro Gly Arg Ser Tyr Ala Ala Asp Ala Gly Tyr Thr Pro-5 1 5 Ala Ala Ala Ala Thr Pro Ala Thr Pro Ala Ala Thr Pro Ala Gly Gly10 15 20 Trp Arg Glu Gly Asp Asp Arg Arg Ala Glu Ala Ala Gly Gly Arg Gln25 30 35 Arg Leu Ala Ser Arg Gln Pro Trp Pro Pro Leu Pro Thr Pro Leu Arg40 45 50 55 Arg Thr Ser Ser Arg Ser Ser Arg Pro Pro Ser Pro Ser Pro ProArg 60 65 70 Ala Ser Ser Pro Thr Ser Ala Ala Lys Ala Pro Gly Leu Ile ProLys 75 80 85 Leu Asp Thr Ala Tyr Asp Val Ala Tyr Lys Ala Ala Glu Ala HisPro 90 95 100 Arg Gly Gln Val Arg Arg Leu Arg His Cys Pro His Arg SerLeu Arg 105 110 115 Val Ile Ala Gly Ala Leu Glu Val His Ala Val Lys ProAla Thr Glu 120 125 130 135 Glu Val Leu Ala Ala Lys Ile Pro Thr Gly GluLeu Gln Ile Val Asp 140 145 150 Lys Ile Asp Ala Ala Phe Lys Ile Ala AlaThr Ala Ala Asn Ala Ala 155 160 165 Pro Thr Asn Asp Lys Phe Thr Val PheGlu Ser Ala Phe Asn Lys Ala 170 175 180 Leu Asn Glu Cys Thr Gly Gly AlaMet Arg Pro Thr Ser Ser Ser Pro 185 190 195 Pro Ser Arg Pro Arg Ser SerArg Pro Thr Pro Pro Pro Ser Pro Ala 200 205 210 215 Ala Pro Glu Val LysTyr Ala Val Phe Glu Ala Ala Leu Thr Lys Ala 220 225 230 Ile Thr Ala MetThr Gln Ala Gln Lys Ala Gly Lys Pro Ala Ala Ala 235 240 245 Ala Ala ThrAla Ala Ala Thr Val Ala Thr Ala Ala Ala Thr Ala Ala 250 255 260 Ala ValLeu Pro Pro Pro Leu Leu Val Val Gln Ser Leu Ile Ser Leu 265 270 275 LeuIle Tyr Tyr 280 756 base pairs nucleic acid single linear cDNA to mRNALolium perenne CDS 3..437 3 AC AAT GAG GAG CCT ATC GCA CCC TAC CAC TTCGAC CTC TCG GGC CAC 47 Asn Glu Glu Pro Ile Ala Pro Tyr His Phe Asp LeuSer Gly His 1 5 10 15 GCA TTC GGG TCC ATG GCG AAG AAG GGC GAG GAG CAGAAG CTC CGC AGC 95 Ala Phe Gly Ser Met Ala Lys Lys Gly Glu Glu Gln LysLeu Arg Ser 20 25 30 GCC GGC GAG CTG GAG CTC CAG TTC AGG CGG GTC AAG TGCAAG TAC CCG 143 Ala Gly Glu Leu Glu Leu Gln Phe Arg Arg Val Lys Cys LysTyr Pro 35 40 45 GAC GGC ACC AAG CCG ACA TTC CAC GTC GAG AAG GGT TCC AACCCC AAC 191 Asp Gly Thr Lys Pro Thr Phe His Val Glu Lys Gly Ser Asn ProAsn 50 55 60 TAC CTG GCT ATT CTG GTG AAG TAC GTC GAC GGC GAC GGC GAC GTGGTG 239 Tyr Leu Ala Ile Leu Val Lys Tyr Val Asp Gly Asp Gly Asp Val Val65 70 75 GCC GTG GAC ATC AAG GAG AAG GGC AAG GAT AAG TGG ATC GAG CTC AAG287 Ala Val Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Glu Leu Lys 8085 90 95 GAG TCG TGG GGA GCA GTC TGG AGG ATC GAC ACC CCC GAT AAG CTG ACG335 Glu Ser Trp Gly Ala Val Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr 100105 110 GGC CCA TTC ACC GTC CGC TAC ACC ACC GAG GGC GGC ACC AAA TCC GAA383 Gly Pro Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr Lys Ser Glu 115120 125 GTC GAG GAT GTC ATT CCT GAG GGC TGG AAG GCC GAC ACC TCC TAC TCG431 Val Glu Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ser Tyr Ser 130135 140 GCC AAG TGAGCAAGAA GTGGAGTGAT CTTCTTCCAA TCAGCTTAAT TTTGACTCAA487 Ala Lys 145 GATCTCAAAT AATCCAGCCG CACATATATA CGAGGCGGTG AGACATACAAGCTCCTCCAT 547 GAGTATATTC ATTCATGCCG TATAGAGAGG AGAAAGATGC CTGAATAAGAGTTTGAGGTC 607 GACACCTTGT GAGAAGTGTA TATAGGAGGA ACCCAATCTG GCTCCATCTTTCTTTGCTCG 667 CACGGTGTAC TGCTAAGGTT ATCTTCTAAC AGGCCAGATT AACCTACTATCTAATATATG 727 CAACGTATGG TCATTTTCCC TAAAAAAAA 756 145 amino acids aminoacid linear protein not provided 4 Asn Glu Glu Pro Ile Ala Pro Tyr HisPhe Asp Leu Ser Gly His Ala 1 5 10 15 Phe Gly Ser Met Ala Lys Lys GlyGlu Glu Gln Lys Leu Arg Ser Ala 20 25 30 Gly Glu Leu Glu Leu Gln Phe ArgArg Val Lys Cys Lys Tyr Pro Asp 35 40 45 Gly Thr Lys Pro Thr Phe His ValGlu Lys Gly Ser Asn Pro Asn Tyr 50 55 60 Leu Ala Ile Leu Val Lys Tyr ValAsp Gly Asp Gly Asp Val Val Ala 65 70 75 80 Val Asp Ile Lys Glu Lys GlyLys Asp Lys Trp Ile Glu Leu Lys Glu 85 90 95 Ser Trp Gly Ala Val Trp ArgIle Asp Thr Pro Asp Lys Leu Thr Gly 100 105 110 Pro Phe Thr Val Arg TyrThr Thr Glu Gly Gly Thr Lys Ser Glu Val 115 120 125 Glu Asp Val Ile ProGlu Gly Trp Lys Ala Asp Thr Ser Tyr Ser Ala 130 135 140 Lys 145 810 basepairs nucleic acid single linear cDNA Lolium perenne CDS 16..804mat_peptide 85..804 5 CAAATTCAAG ACAAG ATG GCG TCC TCC TCG TCG GTG CTCCTG GTG GTG GCG 51 Met Ala Ser Ser Ser Ser Val Leu Leu Val Val Ala -23-20 -15 CTG TTC GCC GTG TTC CTG GGC AGC GCG CAT GGC ATC GCG AAG GTA CCA99 Leu Phe Ala Val Phe Leu Gly Ser Ala His Gly Ile Ala Lys Val Pro -10-5 1 5 CCG GGC CCC AAC ATC ACG GCC GAG TAC GGC GAC AAG TGG CTG GAC GCG147 Pro Gly Pro Asn Ile Thr Ala Glu Tyr Gly Asp Lys Trp Leu Asp Ala 1015 20 AAG AGC ACC TGG TAT GGC AAG CCG ACC GGC GCC GGT CCC AAG GAC AAC195 Lys Ser Thr Trp Tyr Gly Lys Pro Thr Gly Ala Gly Pro Lys Asp Asn 2530 35 GGC GGC GCG TGC GGG TAC AAG GAC GTT GAC AAG GCG CCG TTC AAC GGC243 Gly Gly Ala Cys Gly Tyr Lys Asp Val Asp Lys Ala Pro Phe Asn Gly 4045 50 ATG ACC GGC TGC GGC AAC ACC CCC ATC TTC AAG GAC GGC CGT GGC TGC291 Met Thr Gly Cys Gly Asn Thr Pro Ile Phe Lys Asp Gly Arg Gly Cys 5560 65 GGC TCC TGC TTC GAG ATC AAG TGC ACC AAG CCC GAG TCC TGC TCC GGC339 Gly Ser Cys Phe Glu Ile Lys Cys Thr Lys Pro Glu Ser Cys Ser Gly 7075 80 85 GAG GCT GTC ACC GTC ACA ATC ACC GAC GAC AAC GAG GAG CCC ATC GCA387 Glu Ala Val Thr Val Thr Ile Thr Asp Asp Asn Glu Glu Pro Ile Ala 9095 100 CCC TAC CAT TTC GAC CTC TCG GGC CAC GCG TTC GGG TCC ATG GCG AAG435 Pro Tyr His Phe Asp Leu Ser Gly His Ala Phe Gly Ser Met Ala Lys 105110 115 AAG GGC GAG GAG CAG AAG CTC CGC AGC GCC GGC GAG CTG GAG CTC CAG483 Lys Gly Glu Glu Gln Lys Leu Arg Ser Ala Gly Glu Leu Glu Leu Gln 120125 130 TTC AGG CGG GTC AAG TGC AAG TAC CCG GAC GGC ACC AAG CCG ACA TTC531 Phe Arg Arg Val Lys Cys Lys Tyr Pro Asp Gly Thr Lys Pro Thr Phe 135140 145 CAC GTC GAG AAG GCT TCC AAC CCC AAC TAC CTC GCT ATT CTG GTG AAG579 His Val Glu Lys Ala Ser Asn Pro Asn Tyr Leu Ala Ile Leu Val Lys 150155 160 165 TAC GTC GAC GGC GAC GGT GAC GTG GTG GCG GTG GAC ATC AAG GAGAAG 627 Tyr Val Asp Gly Asp Gly Asp Val Val Ala Val Asp Ile Lys Glu Lys170 175 180 GGC AAG GAT AAG TGG ATC GAG CTC AAG GAG TCG TGG GGA GCA GTCTGG 675 Gly Lys Asp Lys Trp Ile Glu Leu Lys Glu Ser Trp Gly Ala Val Trp185 190 195 AGG ATC GAC ACC CCC GAT AAG CTG ACG GGC CCA TTC ACC GTC CGCTAC 723 Arg Ile Asp Thr Pro Asp Lys Leu Thr Gly Pro Phe Thr Val Arg Tyr200 205 210 ACC ACC GAG GGC GGC ACC AAA TCC GAA GTC GAG GAT GTC ATC CCTGAG 771 Thr Thr Glu Gly Gly Thr Lys Ser Glu Val Glu Asp Val Ile Pro Glu215 220 225 GGC TGG AAG GCC GAC ACC TCC TAC TCG GCC AAG TGAGCA 810 GlyTrp Lys Ala Asp Thr Ser Tyr Ser Ala Lys 230 235 240 263 amino acidsamino acid linear protein not provided 6 Met Ala Ser Ser Ser Ser Val LeuLeu Val Val Ala Leu Phe Ala Val -23 -20 -15 -10 Phe Leu Gly Ser Ala HisGly Ile Ala Lys Val Pro Pro Gly Pro Asn -5 1 5 Ile Thr Ala Glu Tyr GlyAsp Lys Trp Leu Asp Ala Lys Ser Thr Trp 10 15 20 25 Tyr Gly Lys Pro ThrGly Ala Gly Pro Lys Asp Asn Gly Gly Ala Cys 30 35 40 Gly Tyr Lys Asp ValAsp Lys Ala Pro Phe Asn Gly Met Thr Gly Cys 45 50 55 Gly Asn Thr Pro IlePhe Lys Asp Gly Arg Gly Cys Gly Ser Cys Phe 60 65 70 Glu Ile Lys Cys ThrLys Pro Glu Ser Cys Ser Gly Glu Ala Val Thr 75 80 85 Val Thr Ile Thr AspAsp Asn Glu Glu Pro Ile Ala Pro Tyr His Phe 90 95 100 105 Asp Leu SerGly His Ala Phe Gly Ser Met Ala Lys Lys Gly Glu Glu 110 115 120 Gln LysLeu Arg Ser Ala Gly Glu Leu Glu Leu Gln Phe Arg Arg Val 125 130 135 LysCys Lys Tyr Pro Asp Gly Thr Lys Pro Thr Phe His Val Glu Lys 140 145 150Ala Ser Asn Pro Asn Tyr Leu Ala Ile Leu Val Lys Tyr Val Asp Gly 155 160165 Asp Gly Asp Val Val Ala Val Asp Ile Lys Glu Lys Gly Lys Asp Lys 170175 180 185 Trp Ile Glu Leu Lys Glu Ser Trp Gly Ala Val Trp Arg Ile AspThr 190 195 200 Pro Asp Lys Leu Thr Gly Pro Phe Thr Val Arg Tyr Thr ThrGlu Gly 205 210 215 Gly Thr Lys Ser Glu Val Glu Asp Val Ile Pro Glu GlyTrp Lys Ala 220 225 230 Asp Thr Ser Tyr Ser Ala Lys 235 240 30 basepairs nucleic acid single linear not provided 7 GGGTCTAGAG GTACCGTCCGATCGATCATT 30 13 base pairs nucleic acid single linear not provided 8AATGATCGAT GCT 13 20 base pairs nucleic acid single linear not provided9 GGGTCTAGAG GTACCGTCCG 20 26 base pairs nucleic acid single linear notprovided 10 CCCTGCAGAT TATTTGAGAT CTTGAG 26 29 base pairs nucleic acidsingle linear not provided 11 CCCTGCAGTC ATGCTCACTT GGCCGAGTA 29 19 basepairs nucleic acid single linear not provided 12 GAGTACGGCG ACAAGTGGC 1918 base pairs nucleic acid single linear not provided 13 TTCGAGATCAAGTGCACC 18 16 base pairs nucleic acid single linear not provided 14GTGACAGCCT CGCCGG 16 24 base pairs nucleic acid single linear notprovided 15 GGGAATTCCA TGGCGAAGAA GGGC 24 16 base pairs nucleic acidsingle linear not provided 16 GTGCCGTCCG GGTACT 16 16 base pairs nucleicacid single linear not provided 17 CCGTCGACGT ACTTCA 16 19 base pairsnucleic acid single linear not provided 18 GGAGTCGTGG GGAGCAGTC 19 30amino acids amino acid single linear not provided 19 Ile Ala Lys Val XaaPro Gly Xaa Xaa Ile Thr Ala Glu Tyr Gly Asp 1 5 10 15 Lys Trp Leu AspAla Lys Ser Thr Trp Tyr Gly Lys Pro Thr 20 25 30 30 amino acids aminoacid single linear not provided 20 Ile Ala Lys Val Pro Xaa Gly Xaa TrpIle Thr Ala Glu Tyr Gly Asp 1 5 10 15 Lys Trp Leu Asp Ala Lys Xaa ThrXaa Xaa Xaa Xaa Xaa Xaa 20 25 30 30 amino acids amino acid single linearnot provided 21 Ile Ala Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Glu TyrGly Asp 1 5 10 15 Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys ProThr 20 25 30 19 amino acids amino acid single linear not provided 22 AlaAsp Ala Gly Tyr Thr Pro Ala Ala Xaa Xaa Thr Pro Ala Thr Pro 1 5 10 15Ala Xaa Thr 31 amino acids amino acid single linear not provided 23 AlaAla Pro Val Glu Phe Thr Val Glu Lys Gly Ser Asp Glu Lys Asn 1 5 10 15Leu Ala Leu Ser Ile Lys Tyr Asn Lys Glu Gly Asp Ser Met Ala 20 25 30 31amino acids amino acid single linear not provided 24 Ala Ala Pro Val GluPhe Thr Val Glu Lys Gly Ser Asp Glu Lys Asn 1 5 10 15 Leu Ala Leu SerIle Lys Tyr Asn Lys Glu Gly Asp Ser Met Ala 20 25 30 31 amino acidsamino acid single linear not provided 25 Xaa Thr Lys Val Asp Leu Thr ValGlu Lys Gly Ser Asp Ala Lys Thr 1 5 10 15 Leu Val Leu Asn Ile Lys TyrThr Arg Pro Gly Asp Thr Leu Ala 20 25 30 16 amino acids amino acidsingle linear not provided 26 Tyr Thr Thr Glu Gly Gly Thr Lys Ser GluVal Glu Asp Val Ile Pro 1 5 10 15

What is claimed:
 1. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO:
 3. 2. An isolated nucleic acid comprising a nucleotide sequence which encodes at least one antigenic fragment of a ryegrass pollen allergen, wherein said nucleic acid is a portion of the nucleotide sequence of SEQ ID NO:
 3. 3. An isolated nucleic acid encoding a ryegrass pollen allergen comprising the nucleotide sequence of SEQ ID NO: 3 or which differs from a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3 due to degeneracy in the genetic code.
 4. An expression vector comprising a nucleic acid of claim
 1. 5. A host cell containing an expression vector of claim
 4. 6. An expression vector comprising a nucleic acid of claim
 2. 7. A host cell containing an expression vector of claim
 6. 8. The isolated nucleic acid of claim 2, wherein the antigenic fragment comprises at least one T cell epitope recognized by a T cell receptor specific for a ryegrass pollen allergen.
 9. The isolated nucleic acid of claim 2, wherein the antigenic fragment is capable of stimulating T cells specific for a ryegrass pollen allergen.
 10. An expression vector comprising a nucleic acid of claim
 9. 11. A host cell containing an expression vector of claim
 10. 12. An expression vector comprising a nucleic acid of claim
 3. 13. A host cell containing an expression vector of claim
 12. 14. An isolated nucleic acid comprising a nucleotide sequence encoding a protein allergen having at least one T cell epitope recognized by a T cell receptor specific for a T cell epitope containing peptide, wherein said T cell epitope containing peptide is encoded by a nucleic acid which is a portion of the nucleotide sequence of SEQ ID NO:
 3. 15. An isolated nucleic acid comprising a nucleotide sequence encoding a protein allergen capable of stimulating T cells specific for an antigenic fragment of a ryegrass pollen allergen, wherein said antigenic fragment is encoded by a nucleic acid which is a portion of the nucleotide sequence of SEQ ID NO:
 3. 